Profiling and treatment of hypermutant cancer

ABSTRACT

There is provided a method of profiling a tumour, the method comprising determining a relative proportion for each of 96 mutation types, wherein the 96 mutation types are defined as the six possible sequence changes C&gt;A, C&gt;G, C&gt;T, T&gt;A, T&gt;C, or T&gt;G in the context of each of four possible nucleotides (A, C, G, or T) at the position immediately 5′ to the mutation and each of four possible nucleotides at the position immediately 3′ to the mutation; assigning the tumour, using the determined relative proportion for each of the 96 mutation types, to at least one of eight clusters defined herein; and determining at least one tumour characteristic based on the assignment to a cluster.

RELATED APPLICATION

This claims priority from U.S. Provisional Application No. 62/553,375 filed Sep. 1, 2017 and entitled “PROFILING AND TREATMENT OF HYPERMUTANT CANCER”, which is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates generally to tumour profiling and treatment. More specifically, the disclosure relates to the profiling and treatment of hypermutant cancer.

BACKGROUND

Neoplastic cells circumvent the usual DNA repair safeguards, allowing for the accumulation of mutations that drive cancer. Yet, even a cancer cell's mutation burden is typically kept within specific bounds.

Recently, a group of cancers with high mutation burden (hypermutation) has been reported (Shlien, et al Nat Genet 2015). Patients with these cancers often have very different outcomes than those with the same tumour type lacking a high mutation burden, and are the only group responsive to immune checkpoint inhibition (Bouffet et al J Clin Onc 2016).

However, due to limited sample sizes, the threshold and extent of hypermutation across cancer, as well as the exact tumour types involved, are currently unknown.

Furthermore, since different tumour types with hypermutation have never been considered together before, it is not currently known if their hypermutation arises via similar molecular mechanisms, or at the same time during tumour evolution.

It would be desirable to provide a means of profiling hypermutant cancers.

It would be desirable to provide profiles indicative of tumour biology.

It would be desirable to establish treatments for hypermutant cancers.

SUMMARY

It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous approaches.

In a first aspect, the present disclosure provides a method of profiling a tumour, the method comprising sequencing nucleic acid from a sample obtained from the tumour; identifying mutations relative to a reference sequence, wherein a mutation is defined with respect to the pyrimidine of a base pair; determining a relative proportion for each of 96 mutation types, wherein the 96 mutation types are defined as each of six possible pyrimidine base changes C>A, C>G, C>T, T>A, T>C, or T>G in the context of each of four possible nucleotides (A, C, G, or T) at the position immediately 5′ to the mutation and each of four possible nucleotides (A, C, G, or T) at the position immediately 3′ to the mutation; assigning the tumour, using the determined relative proportion for each of the 96 mutation types, to at least one of eight clusters, wherein the at least eight clusters are depicted as 1 to 8 in the heat map of FIG. 19 , wherein each of the clusters is defined by a visual representation the relative proportions of each of the 96 mutation types for the cluster; and determining at least one tumour characteristic based on the assigning.

Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

FIG. 1 (1A to 1E) depicts the landscape of hypermutation across 81,337 pediatric and adult cancers. FIG. 1A) Tumour mutation burden for 2,885 pediatric cancers. Tumours with 10 mut/mb or more are considered hypermutant, tumours with 100 mut/mb are considered ultra-hypermutant. FIG. 1B) Breakdown of hypermutation pediatric cohort by tumour type. Corresponding pie graphs for each mutation range depicts the proportion of tumours harboring mutations in genes implicated in replication repair deficiency (RRD) (MSH2, MLH1, MSH6, PMS2 POLE, POLD1). FIG. 1C) Tumour mutation burden range for 78,452 adult tumours and tumour type breakdown in ultra-hypermutant and hypermutant groups. Tumour types are ranked in descending order by median mutation burden. FIG. 1D) Tumour types that show enrichment for MSI-MSI-H tumours cluster in the 10-100 mut/mb range while tumours with both mismatch repair and polymerase proofreading in the same types occupy the ultra-hypermutant range. FIG. 1E) Pie graphs represent the proportion of ultra-hypermutant, hypermutant and lowly mutated tumours and their correlation with MSI-H and MMR/POL mutations. Acronyms: GI=Gastrointestinal, AML=Acute Myeloid Leukemia, NBL=Neuroblastoma, RMS=Rhabdomyosarcoma, STS=Soft tissue sarcoma, OS=Osteosarcoma, EWS=Ewing's sarcoma, WLMS=Wilm's tumour, RCC=Renal cell carcinoma, NP&PNS=nasopharynx and paranasal sinuses undifferentiated carcinoma, MM&MDS=Bone marrow myelodysplastic syndrome

FIG. 2 (2A to 2D) depicts characterization of known and novel POLE and POLD1 driver mutations FIG. 2A) Examples of tumours with three or more POLE/POLD1 mutations and the corresponding number of tumours found in the entire dataset harboring those mutations (grey bars). The height of bars corresponds to mutation burden of other tumours with that mutation. No bars indicates no other tumours were identified with this mutation. One clear driver emerges in each case. FIG. 2B) Graphical representation of all drivers (top) and passengers (bottom) identified in POLE (left panel) and POLD1 (right panel) following in-vivo mutagenesis screening criteria. Circles (originally coloured green) represent previously known drivers while other circles (originally coloured yellow) represent novel drivers, first described here. FIG. 2C) Codons in POLE at which driver mutations are found and whether they are sensitive to amino acid changes. Invariable codons represent codons at which only one amino acid change was detected. Insensitive codons represent codons at which mutation burden was high, regardless of amino acid change. Sensitive codons represent codons at which certain amino acid changes would abrogate the mutator effect. Bars (originally shaded green, yellow, and red) represent strong, moderate and weak mutation burden phenotypes, respectively. FIG. 2D) All tumours harboring POLE and POLD1 tumours, by type, colored by whether the mutation was a driver or passenger.

FIG. 3 (3A to 3C) depicts unsupervised clustering by trinucleotide context reveals mutational etiology of hypermutant tumours. FIG. 3A) Top panel: Unsupervised hierarchical clustering of 1521 tumours by trinucleotide context reveals 8 distinct clusters (colored dendrogram). Middle: Disease type, MSI status. Bottom panel: Heatmap colored by proportion of mutations from each class of mutational signatures. FIG. 3B) Top: Range of tumours types found in clusters C1, C2 and C3, size of circle indicates number of tumours. Middle: Box plots displaying mutation burden (mut/MB) in each of C1, C2, C3. Bottom: Pie charts displaying proportion of tumours in each cluster, which are MSI-High, POLE-mutant and children respectively. FIG. 3C) Average proportion of mutations attributed by 4 mutational signature classes, Tobacco Smoke (Signature 4), Alkylating agents (Signature 11), UV Light (Signature 7) and APOBEC (Signature 2 and 13). Color of circles indicates cluster tumours belong to, size indicates number of tumours in this cluster and tumour type and y axis indicates average proportion of mutations attributed to each signature.

FIG. 4 (4A to 4F) depicts mutational context in hypermutant tumours determined by timing and etiology of mutation. FIG. 4A) Left: Average proportion of mutations by trinucleotide context from panel sequencing. Germline status and treatment history unknown. N indicates number of tumour samples used to calculate average proportions. Right: Average proportion of mutations by trinucleotide context in exomes with known germline status/treatment history. N indicates number of tumour samples used to calculate average proportions. FIG. 4B) Example mutational signatures in exomes from tumours with known germline status/treatment history. FIG. 4C-F) Examples of subclonal mutational signatures determined from allelic read depth calculations on panel sequencing data. N indicates number of mutations in cluster. Signatures 21, 6, 15, and 14 are those previously associated with MMR deficiency, Signature 10 was previously associated with POLE mutations, and Signature 11 was previously associated with treatment with alkylating agents. FIG. 4C) Subclonal mutational signatures in an adult colorectal carcinoma with somatic POLE mutation reveal early mutations due to POLE mutations and later mutations due to POLE mutation and mismatch repair defiance FIG. 4D) Subclonal mutational signatures in a pediatric glioblastoma reveal early mutations due to deficits in mismatch repair and later mutations due to mismatch repair and POLE proofreading mutation FIG. 4E) Mutational signatures present in subclonal clusters of mutations in a lung adenocarcinoma reveal early mutations due to smoking and later mutations due to replication repair deficiency. FIG. 4F) Mutational signatures present in 3 subclonal clusters of mutations in a skin melanoma reveal early mutations due to primarily UV light exposure and later mutations due mostly to exposure to alkylating agents.

FIGS. 5 (5A and 5B) depicts that high mutation burden and specific signatures reveal predisposition and treatment history in pediatric cancers. FIG. 5A) Signature analysis for all ultra-hypermutant (>100 mut/mb) pediatric tumours. All signatures associated with replication repair deficiency were combined (black). Signatures associated with alkylating agents are shown (originally coloured turquoise). 19 patients with ultra-hypermutant tumours were confirmed for germline cancer predisposition involving replication repair genes. Numbers above each bar represent the mutation burden for that tumour. FIG. 5B) Signature analysis for hypermutant pediatric tumours (10-100 mut/mb)-hypermutant pediatric tumours of the three tumour types typically associated with CMMRD (brain, GI, leukemia/lymphoma) were enriched for RRD signatures. Two brain tumour patients were retrospectively confirmed as CMMRD (50 mut/mb) and Lynch (34 mut/mb) respectively.

FIGS. 6 (6A and 6B) shows that targeted panel sequencing accurately predicts mutation burden determined by exome or whole genome sequencing. FIG. 6A) Linear correlation between tumour mutation burden derived from whole exome sequencing (WES) and tumour mutation burden derived from 1.1-3.25 MB of targeted panel sequencing (R-squared=0.94). FIG. 6B) Tumour mutation burden on a test dataset of 14 tumours that underwent targeted panel sequencing, whole exome sequencing, and whole genome sequencing.

FIG. 7 shows determination of a hypermutation threshold in human cancer through segmented linear regression analysis. Left panel: Pediatric cohort of 2,885 tumours ranked by mutation burden (mutations/Mb). Right panel: Adult cohort of 78,452 tumours ranked by mutations burden (mutations/Mb). Dotted lines indicate segment boundaries determined by segmented linear regression analysis using an iterative method, selecting points at which there was a significant change in the slope of the linear regression. Grey solid lines (originally coloured red) indicate linear regression lines of individual segments. The boundary points at which there was observed to be a significantly large gain in the slope of the regression line, were selected as the thresholds of hypermutation (9.91 and 9 for pediatric and adult cohort respectively), which were rounded to 10 for the remainder of the analyses described in this study.

FIG. 8 shows that a large range of tumour types exhibit microsatellite instability. 81 unique tumour histologies with at least 1 MSI-H tumour.

FIG. 9 shows that pediatric cancers with a combined mismatch repair deficiency and polymerase driver mutation display an ultra-hypermutant phenotype. Cohort of 2885 pediatric tumours harboring either MMR and polymerase mutations concurrently, only mismatch repair mutations, only polymerase mutations, or neither. The presence of a mismatch repair mutation is significantly associated with hypermutation, while a polymerase mutation alone is not.

FIG. 10 shows that L424I and L424V Pol ε mutations vary in their degree of reduced exonuclease function. Excision rate constants of 3′-5′ exonuclease activity catalyzed by Pol ε and the indicated mutants at 37° C.

FIGS. 11 (11A to 11C) depicts signature analysis of hypermutant lung cancer reveals predominance of Signature 7 (UV Light) in hypermutant lung squamous cell carcinoma. FIG. 11A) Proportion of Signature 7 versus Signature 4 (Smoking) in 100 hypermutant lung cancer samples run on cancer panel. Circle sizes indicate tumour mutation burden in mut/mb, and colors represent lung cancer subtype (i.e. adenocarcinoma, squamous cell carcinoma, non-small cell (not otherwise specified) and other. FIG. 11B) Proportion of Signature 4 in lung cancers by subtype. FIG. 11C) Proportion of Signature 7 in lung cancers by subtype.

FIG. 12 shows that hypermutant soft tissue sarcomas from TCGA exhibit signature of SNVs consistent with UV light exposure. Number of somatic SNVs in 103 TCGA sarcoma exomes (minimum 50 somatic SNVs). Magenta indicates proportion of mutations explained by Signature 7 (UV light). Lighter grey indicates proportion of mutations explained by all other signatures.

FIGS. 13 (13A to 13C) shows that clustering identifies tumours with differences in evolutionary dynamics and survival. FIG. 13A) Histogram displaying number of SNVs by variant allele fraction (VAF) in each of the 8 major clusters identified by hierarchal clustering in FIG. 3 . Colors indicate type of SNV (Stopgain, missense and synonymous mutations). FIG. 13B) VAF vs median cumulative mutation burden plotted for each of the 3 replication repair clusters. Cluster 1 tumours exhibit an early burst of mutations (around 0.4 VAF) with a second burst of mutations later in tumour evolution (around 0.2 VAF). Cluster 3 tumours display a single burst of mutations around 0.2 VAF, and Cluster 2 tumours exhibit a more gradual accumulation of mutations throughout their evolution. FIG. 13C) Kaplan-Meier plot of overall survival for tumours with mutational signatures consistent with clusters 1, 2, or 3. Cluster 3, n=27. Cluster 2, n=168. Cluster 1, n=22. P<0.0001.

FIG. 14 shows procedure for diagnosing cancer predisposition syndrome via tumour-specific panel sequencing. Panel sequencing results displaying high tumour mutation burden (TMB), a driver mutation in POLE or POLD1 and signatures reflective of replication repair deficiency are highly specific for a Biallelic Mismatch Repair Deficiency diagnosis. Clinical intervention includes surveillance protocols and immune checkpoint inhibition therapy for active tumours.

FIG. 15 depicts, in the left panel, 15 patients for which only panel sequencing was performed prior to confirmation of predisposition syndrome diagnosis. Darkest squares indicate signatures corresponding to MMR and the subsequent identification of a germline mutation in an MMR gene. Lightest grey squares indicate the same for POLE. In the right panel is shown an example of a glioma found via surveillance. In the bottom right panel is shown a colorectal cancer responding to Anti-PD1 therapy following confirmation of germline MMR mutation.

FIG. 16 depicts a barplot showing the percentage of MSI-H tumours out of all tumours at the respective mutation burden bin. MSI-H tumours concentrated in the 10-100 mutation burden range.

FIG. 17 depicts a boxplot showing the variant allele fraction of POLE R446Q mutation in 64 tumours. The majority of mutations clustered at 0.5, signifying that it is a germline SNP and not a hypermutation driver.

FIG. 18 shows that mutations in replication repair genes are randomly distributed and do not cluster around the hypermutation threshold further supporting that these specific alterations are passengers and not driving hypermutation.

FIG. 19 depicts a heat map providing a visual representation of the relative proportions of each of the 96 mutation types for each of the clusters 1 to 8.

DETAILED DESCRIPTION

Generally, the present disclosure provides a method of profiling a tumour, the method comprising determining a relative proportion for each of 96 mutation types for the tumour, wherein the 96 mutation types are defined as the six possible sequence changes C>A, C>G, C>T, T>A, T>C, or T>G in the context of each of four possible nucleotides (A, C, G, or T) at the position immediately 5′ to the mutation and each of four possible nucleotides at the position immediately 3′ to the mutation; assigning the tumour, using the determined relative proportion for each of the 96 mutation types, to one of at least eight clusters defined herein; and determining at least one tumour characteristic based on the assignment to a cluster.

Methods of Tumour Profiling

In one aspect, there is provided a method of profiling a tumour, the method comprising sequencing nucleic acid from a sample obtained from the tumour; identifying mutations relative to a reference sequence, wherein a mutation is defined with respect to the pyrimidine of a base pair; determining a relative proportion for each of 96 mutation types, wherein the 96 mutation types are defined as each of six possible pyrimidine base changes C>A, C>G, C>T, T>A, T>C, or T>G in the context of each of four possible nucleotides (A, C, G, or T) at the position immediately 5′ to the mutation and each of four possible nucleotides (A, C, G, or T) at the position immediately 3′ to the mutation, assigning the tumour, using the determined relative proportion for each of the 96 mutation types, to at least one of eight clusters, wherein the at least eight clusters are depicted as 1 to 8 in the heat map of FIG. 19 , wherein each of the clusters is defined by a visual representation of the relative proportions of each of the 96 mutation types for the cluster; and determining at least one tumour characteristic based on the based on the assigning.

The shading of the heat map depicted in FIG. 19 corresponds to the numerical values for the mean proportion for each mutation type for each cluster, 1 to 8, as shown in Table 1, wherein “Mut” is indicative of the mutation, “5′” is indicative of the 5′ base, and “3′” is indicative of the 3′ base.

TABLE 1 Mean Proportion of Mutation Types for Clusters 1 to 8 Mean Proportion for Clusters Mut 5′ 3′ 1 2 3 4 5 6 7 8 C > A A A 6.7E−05 0.001303 0.00119 0.017772 0.001499 0.002243 0.002892 0.019454 C > A A C 0.002389 0.003823 0.003755 0.026454 0.002257 0.001905 0.004423 0.026279 C > A A G 0 0.00119 0.000792 0.017313 0.000452 0.000727 0.001758 0.014491 C > A A T 0.009421 0.002499 0.006822 0.016983 0.000599 0.000833 0.001252 0.014017 C > A C A 0.003067 0.00708 0.003819 0.05069 0.001325 0.003465 0.003623 0.048299 C > A C C 0.004592 0.011953 0.002766 0.05509 0.001926 0.002554 0.0033 0.045063 C > A C G 0.00161 0.007254 0.002327 0.034315 0.001284 0.001854 0.002787 0.030886 C > A C T 0.037945 0.029925 0.014682 0.036224 0.000878 0.00349 0.003352 0.029591 C > A G A 0.001125 0.002937 0.002638 0.021099 0.001394 0.001877 0.00328 0.022232 C > A G C 0.005352 0.004551 0.002743 0.032029 0.001812 0.001296 0.003621 0.024923 C > A G G 0.001282 0.002862 0.00103 0.023932 0.000308 0.00086 0.001465 0.021978 C > A G T 0.032471 0.008021 0.008659 0.014444 0.002057 0.001507 0.002714 0.016893 C > A T A 0.002468 0.002416 0.030984 0.02373 0.000599 0.002695 0.023976 0.028258 C > A T C 0.006074 0.004215 0.008444 0.038228 0.001522 0.004612 0.015313 0.037669 C > A T G 0.000658 0.001553 0.003072 0.012278 0.000797 0.001133 0.009061 0.01494 C > A T T 0.041834 0.006319 0.190779 0.03142 0.001709 0.002654 0.015214 0.027702 C > G A A 0.000569 0.001268 0.000741 0.006863 0.000744 0.000844 0.003019 0.00775 C > G A C 0.000114 0.002475 0.000842 0.004127 0.001494 0.001292 0.003434 0.004868 C > G A G 0.000251 0.001332 0.000468 0.003539 0.000665 0.00116 0.001847 0.003259 C > G A T 0 0.001943 0.000872 0.004021 0.000726 0.001104 0.002654 0.005493 C > G C A 0.00032 0.00168 0.00023 0.008079 0.000856 0.001644 0.00432 0.007216 C > G C C 0.000559 0.002767 0.001219 0.011364 0.001386 0.002353 0.004633 0.006497 C > G C G 0.000379 0.003067 0.000998 0.012243 0.0013 0.002673 0.004404 0.007152 C > G C T 0.000202 0.001803 0.001843 0.006568 0.001703 0.00159 0.004905 0.005911 C > G G A 0.000238 0.001606 0.000929 0.005728 0.000382 0.000724 0.001759 0.00454 C > G G C 0.000404 0.003352 0.001803 0.006832 0.001102 0.001522 0.002245 0.008064 C > G G G 5.71E−05 0.001414 0.00021 0.005349 0.000993 0.001158 0.002204 0.005642 C > G G T 7.05E−05 0.001991 0.001271 0.003366 0.001426 0.000885 0.004135 0.005597 C > G T A 0 0.001682 0.000244 0.01032 0.000721 0.002827 0.088732 0.026979 C > G T C 0.000343 0.002414 0.000753 0.01316 0.000957 0.003088 0.027984 0.016404 C > G T G 0 0.001217 0.000503 0.004416 0.000578 0.001376 0.014448 0.003659 C > G T T 0.001043 0.004031 0.000942 0.013253 0.00256 0.004521 0.105522 0.03456 C > T A A 0.009098 0.015807 0.002356 0.012255 0.018988 0.003111 0.007497 0.008034 C > T A C 0.021673 0.014512 0.007633 0.008304 0.107317 0.028438 0.006307 0.006785 C > T A G 0.080822 0.098997 0.029695 0.024394 0.018757 0.009504 0.01971 0.008963 C > T A T 0.01273 0.008974 0.006131 0.00745 0.061462 0.008133 0.004222 0.006623 C > T C A 0.005011 0.009953 0.002726 0.016459 0.026098 0.069057 0.015157 0.018233 C > T C C 0.013316 0.012312 0.003786 0.016002 0.119295 0.116254 0.010024 0.015242 C > T C G 0.075944 0.103416 0.024621 0.028195 0.024174 0.052378 0.031736 0.020032 C > T C T 0.013479 0.012866 0.006972 0.016392 0.07329 0.07903 0.012472 0.018323 C > T G A 0.024254 0.028917 0.005288 0.011127 0.024443 0.00376 0.01157 0.008069 C > T G C 0.059279 0.046541 0.024879 0.014863 0.093441 0.027676 0.009702 0.01053 C > T G G 0.222447 0.148718 0.070994 0.015991 0.02658 0.007628 0.022776 0.012521 C > T G T 0.038951 0.030229 0.021253 0.011066 0.064453 0.013225 0.009746 0.007571 C > T T A 0.007213 0.010064 0.004939 0.014983 0.023016 0.072722 0.171925 0.041898 C > T T C 0.028449 0.011086 0.016469 0.010613 0.122656 0.197681 0.05397 0.019335 C > T T G 0.078732 0.052801 0.219143 0.014147 0.01483 0.08704 0.065382 0.021316 C > T T T 0.027756 0.008177 0.02943 0.012661 0.078928 0.065168 0.077588 0.027306 T > A A A 0.000295 0.001176 0.000209 0.002711 0.00066 0.001701 0.001204 0.002119 T > A A C 0.001226 0.003815 0.000852 0.005563 0.001046 0.001309 0.001153 0.002691 T > A A G 0.0004 0.001401 0.000458 0.008168 0.000793 0.00164 0.002408 0.008367 T > A A T 0.001352 0.002917 0.001796 0.002225 0.000307 0.00301 0.000762 0.00305 T > A C A 0 0.000781 0.000352 0.005749 0.000273 0.000998 0.000917 0.003744 T > A C C 0.000369 0.002485 0.001001 0.008503 0.000572 0.001611 0.002477 0.006996 T > A C G 0.0005 0.004052 0.001149 0.019982 0.001139 0.001982 0.003061 0.019045 T > A C T 0.000809 0.001423 0.000821 0.006551 0.000594 0.002184 0.001376 0.006358 T > A G A 0 0.0012 0.000226 0.003155 0.000839 0.001257 0.000893 0.006023 T > A G C 0.002031 0.002942 0.000182 0.003327 0.000677 0.000932 0.000955 0.002762 T > A G G 0.00019 0.003729 0.000445 0.008214 0.001309 0.001757 0.001271 0.00739 T > A G T 0.000321 0.00107 0.000202 0.00161 0.00086 0.001172 0.001481 0.00341 T > A T A 0 0.000781 0.000138 0.003102 0.000457 0.001356 0.00024 0.003029 T > A T C 0.001246 0.00113 0.000725 0.003455 0.000207 0.002091 0.000854 0.004073 T > A T G 0.000229 0.000808 0.00015 0.005573 0.000305 0.001333 0.000518 0.005702 T > A T T 0.000267 0.000915 0.00279 0.002261 0.000528 0.004013 0.001072 0.003578 T > C A A 0.006228 0.010066 0.004417 0.006428 0.002687 0.002514 0.004557 0.004975 T > C A C 0.005627 0.00713 0.004952 0.003248 0.002543 0.001447 0.001645 0.0029 T > C A G 0.004509 0.021637 0.005165 0.008724 0.003567 0.003323 0.009699 0.007189 T > C A T 0.00324 0.007429 0.004211 0.003789 0.002231 0.002848 0.005327 0.003113 T > C C A 0.001378 0.007187 0.000923 0.002191 0.001319 0.001957 0.001896 0.003062 T > C C C 0.004677 0.009799 0.003691 0.00541 0.002954 0.002671 0.003789 0.00364 T > C C G 0.003798 0.025847 0.003957 0.011165 0.002454 0.003085 0.004987 0.010078 T > C C T 0.002998 0.009297 0.003321 0.005057 0.002592 0.004473 0.003038 0.004469 T > C G A 0.014476 0.017372 0.01155 0.004864 0.003773 0.001411 0.002532 0.003809 T > C G C 0.012038 0.011524 0.015514 0.00185 0.004029 0.001397 0.002503 0.0027 T > C G G 0.012999 0.020376 0.008717 0.005925 0.004941 0.003671 0.006668 0.00738 T > C G T 0.008026 0.011538 0.012768 0.005125 0.003119 0.007672 0.003825 0.003653 T > C T A 0.002157 0.00638 0.002805 0.004462 0.00213 0.003264 0.002 0.001159 T > C T C 0.003458 0.007139 0.00666 0.003498 0.002031 0.002641 0.002223 0.001386 T > C T G 0.002926 0.009561 0.005562 0.004882 0.001529 0.002926 0.001109 0.00204 T > C T T 0.002948 0.005824 0.005819 0.002753 0.001366 0.002989 0.001503 0.000691 T > G A A 6.7E−05 0.000486 0.001478 0.000183 0.000228 0.000604 0.000496 0.000545 T > G A C 0.001077 0.001094 0.003775 0.000771 8.55E−05 0.000615 0.000651 0.000557 T > G A G 0.000499 0.001114 0.001846 0.000516 0.000706 0.001337 0.001154 0.001011 T > G A T 0.003055 0.00097 0.011338 0.000525 0.000155 0.000611 0.000462 0.000783 T > G C A 0.000196 0.00167 0.002233 0.001327 0.000868 0.000933 0.001278 0.002199 T > G C C 0.000731 0.004141 0.001955 0.001327 0.000715 0.001481 0.001622 0.00119 T > G C G 0.000628 0.00923 0.003836 0.005045 0.000693 0.002199 0.002348 0.004793 T > G C T 0.003131 0.005064 0.015868 0.001173 0.000679 0.001849 0.001671 0.001068 T > G G A 0.000365 0.000912 0.001333 0.002413 0.000422 0.000424 0.000223 0.000884 T > G G C 0.001875 0.001146 0.002466 0.000516 0.000595 0.000523 0.000749 0.00071 T > G G G 0.001543 0.002292 0.00168 0.003201 0.002064 0.002655 0.001429 0.002581 T > G G T 0.003994 0.003071 0.004097 0.001524 0.001203 0.001403 0.001174 0.000958 T > G T A 0 0.000364 0.004756 0.000764 0.000183 0.000486 0.000504 0.000471 T > G T C 0.001569 0.001778 0.006492 0.001079 0.001107 0.00163 0.001786 0.001199 T > G T G 0.000386 0.001434 0.001971 0.002192 0.000242 0.001367 0.001185 0.002327 T > G T T 0.006112 0.001191 0.048665 0.001796 0.000477 0.001973 0.001265 0.001102

Thus, in some embodiments, the relative proportion for each of the 96 mutation types for each cluster is the corresponding numerical value for the mean proportion depicted in Table 1.

It will be understood in the above that the definition of mutations and mutation types by reference to pyrimidines is merely a convention, and the method would inherently encompass methods in which the analogous definitions, types, and cluster heat maps were instead defined with respect to purines.

By “profiling,” is meant determination of at least one tumour characteristic.

By “tumour characteristic” is meant any feature of the tumour, including e.g. aspects of its present biology and/or tumour origin or development. This could include, as non-limiting examples, gene mutations, oncogenic mutations, germline mutations, early mutations, late mutations, tissue origin, mutational history (or a step thereof), order of mutational events, mutagen exposure, order of mutagen exposure, prior treatment, etc. It could also include, e.g., a correlation with histology, biomarker status, tumour grade, tumour staging, tumour metastasis, resistance to a therapeutic, and/or prognosis.

By “assigning” is meant associating the tumour with at least one of the eight clusters. Any suitable means of achieving an assignment may be employed, including the following non-limiting examples. The tumour could be categorized based on the relative prevalence of each of the 96 mutation types. Categories could be established for each of the eight categories base, e.g. on numerical ranges or absolute values plus or minus 5%, 10%, 15%, 20%, 25%, etc. A tumour could be assigned to a cluster based, e.g., on the best categorization. Statistical methods could be employed to assess matching or best fist.

In some embodiments, not all relative proportions need be used for making the assignment of a tumour to a cluster. In some embodiments, the assigning is carried out using the relative proportion of one of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least one of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least two of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least three of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least four of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least five of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least six of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least 12 of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least 24 of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least 36 of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least 48 of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least 60 of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least 72 of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using the relative proportions of at least 84 of the 96 mutation types determined for the tumour. In some embodiments, the assigning is carried out using all 96 of the relative proportions determined for the tumour.

In the above embodiments involving subsets of the 96 mutation types, in some embodiments the relative proportions used in the assigning are the most informative of the relative proportions. By “most informative” of the relative proportions is meant those relative proportions that are most distinguishing. For examples, they may be significantly depleted or enriched in a cluster relative to others.

In some embodiments, the step of assigning comprises assigning to the cluster based on highest cosine similarity to the mean proportion values for the cluster recited in Table 1.

In some of these embodiments, a minimum of 0.75 cosine similarity may be employed as a threshold value for the assignment. In another embodiment, the threshold may be 0.7. In another embodiment, the threshold may be 0.8. In another embodiment, the threshold may be 0.9. In another embodiment, the threshold may be 0.95.

In another embodiment, the assigning could be carried out by clustering. In one embodiment, the assigning may be based on based on distance. For example Levenshtein's distance may be used for the assigning. For example, Euler's distance may be used for the assigning. Other distance metrics may also be used, according to requirements.

In some embodiments, the step of assigning comprises assigning to the cluster based on ranges for the frequencies of each mutation type calculated based on the mean proportions of Table 1 and the corresponding standard deviation values (Std Dev) for clusters 1 to 8 that are depicted in Table 2.

TABLE 2 Standard Deviations for the Mean Proportions for Clusters 1 to 8 Std Dev for Clusters Mut 5′ 3′ 1 2 3 4 5 6 7 8 C > A A A 0.0002595 0.0041439 0.0032608 0.0119057 0.0048422 0.005093 0.0072993 0.0144502 C > A A C 0.0030569 0.0072622 0.0046601 0.0189826 0.0058685 0.0042982 0.0079754 0.0178127 C > A A G 0 0.0042131 0.0024838 0.0114635 0.002336 0.0024988 0.0053597 0.0124883 C > A A T 0.0054846 0.0058485 0.0059137 0.0118644 0.002766 0.0025598 0.0041304 0.0109158 C > A C A 0.0024806 0.0103239 0.00497 0.0214626 0.00375 0.0079367 0.0077395 0.0236659 C > A C C 0.0033922 0.0130088 0.006124 0.0260265 0.0048793 0.0051591 0.0078407 0.0230598 C > A C G 0.0017717 0.009823 0.0038129 0.0185407 0.0032862 0.0038233 0.0070639 0.0162666 C > A C T 0.0205006 0.0239408 0.0141586 0.0201944 0.0023286 0.0056525 0.0069079 0.0194444 C > A G A 0.0013321 0.0061895 0.003773 0.0156266 0.004379 0.0039386 0.0067378 0.0141242 C > A G C 0.0040586 0.0079601 0.0037048 0.0163821 0.0045099 0.0036569 0.0081921 0.0187841 C > A G G 0.0019012 0.0063641 0.0033833 0.0221213 0.0014713 0.0029913 0.0045468 0.0144501 C > A G T 0.0137013 0.0119439 0.007969 0.0112118 0.0045037 0.0036695 0.0061766 0.013116 C > A T A 0.003762 0.0061854 0.019322 0.025547 0.0021934 0.0051413 0.0219588 0.0176898 C > A T C 0.0031478 0.0078095 0.0065275 0.0201077 0.0035549 0.0065461 0.0142899 0.0195741 C > A T G 0.001048 0.0043533 0.0037298 0.0101746 0.0028682 0.0030056 0.0131488 0.0118502 C > A T T 0.0181503 0.0113834 0.0726526 0.0374747 0.0037857 0.0049425 0.0141139 0.0158362 C > G A A 0.0010317 0.0041451 0.0014682 0.0087312 0.0023622 0.0026852 0.0065718 0.0094584 C > G A C 0.0004421 0.0055014 0.0020643 0.0062467 0.0038456 0.0030985 0.0073949 0.007776 C > G A G 0.0006614 0.0045739 0.0023585 0.0057338 0.0025753 0.0032575 0.0057987 0.0055832 C > G A T 0 0.00492 0.002179 0.0058164 0.0028106 0.0032393 0.0063967 0.006244 C > G C A 0.0007 0.0047322 0.0009632 0.0082992 0.0028212 0.0039723 0.0088852 0.0091725 C > G C C 0.0013797 0.0065612 0.0028974 0.0099251 0.0035483 0.004847 0.0085915 0.0068398 C > G C G 0.0007969 0.0062266 0.0028908 0.0111333 0.0032713 0.0047066 0.0078663 0.0085754 C > G C T 0.0005603 0.0048111 0.0030163 0.0084157 0.0049312 0.0037096 0.0081422 0.0075746 C > G G A 0.0006947 0.0045624 0.002038 0.0095707 0.0016727 0.0023972 0.0046031 0.0060832 C > G G C 0.0007638 0.0066215 0.0033749 0.0092563 0.0030308 0.0039541 0.0058586 0.0093659 C > G G G 0.0002211 0.0040916 0.0008422 0.0071569 0.0032841 0.0033122 0.0066091 0.0069762 C > G G T 0.0002729 0.0049018 0.0022559 0.0066613 0.0057428 0.0029432 0.0075003 0.0069928 C > G T A 0 0.0055823 0.0008283 0.0142574 0.0039999 0.0084125 0.0417002 0.0360654 C > G T C 0.000751 0.0056184 0.0024403 0.0119991 0.0028617 0.0057843 0.0211052 0.0113682 C > G T G 0 0.003799 0.0021326 0.0066008 0.0024752 0.0035604 0.0149763 0.0062463 C > G T T 0.0015205 0.0085396 0.0025392 0.0121808 0.0052453 0.0090779 0.0467566 0.0550912 C > T A A 0.0066265 0.0157468 0.0030307 0.0117878 0.0115829 0.0055374 0.0104628 0.0091602 C > T A C 0.0129305 0.0142151 0.0065761 0.0081682 0.0317933 0.015375 0.0106645 0.0087731 C > T A G 0.0247868 0.05085 0.0181394 0.01494 0.0168833 0.0100254 0.0180587 0.0083301 C > T A T 0.0057859 0.0106363 0.0068695 0.00802 0.0227499 0.0087477 0.0076722 0.0079624 C > T C A 0.0033377 0.0114202 0.003669 0.0144497 0.0150796 0.0228448 0.015854 0.0122498 C > T C C 0.0080867 0.0125512 0.0053586 0.0158169 0.041537 0.0367514 0.0131713 0.0128216 C > T C G 0.0202904 0.0507454 0.0171792 0.0211806 0.0192641 0.0244128 0.0214387 0.0150531 C > T C T 0.0065427 0.0140946 0.0064929 0.0145021 0.0259832 0.0276278 0.0152985 0.0109122 C > T G A 0.0124183 0.0210376 0.0056939 0.010441 0.0141201 0.006271 0.0124556 0.0090126 C > T G C 0.0163492 0.0283639 0.0125448 0.0130065 0.039711 0.0162894 0.0109415 0.009643 C > T G G 0.04068 0.0703008 0.0401502 0.0104244 0.0202383 0.0096827 0.0197295 0.0096317 C > T G T 0.0086611 0.0220909 0.0095118 0.0098223 0.0261977 0.0107361 0.0125057 0.0103458 C > T T A 0.003614 0.0148287 0.0059801 0.0121183 0.0138185 0.0323985 0.0621188 0.0285873 C > T T C 0.0102873 0.0124219 0.00927 0.0106852 0.0350252 0.0451308 0.029443 0.0139328 C > T T G 0.0278252 0.0308167 0.072775 0.0135731 0.0113412 0.036986 0.0314477 0.0195188 C > T T T 0.010273 0.0118584 0.0107877 0.0134295 0.0274868 0.0232987 0.0368886 0.017879 T > A A A 0.0007871 0.0041769 0.0008831 0.0054006 0.0024975 0.0038955 0.0049019 0.0047918 T > A A C 0.0034941 0.0069411 0.0017901 0.007299 0.0027845 0.0035111 0.0038967 0.0050771 T > A A G 0.0010935 0.0042195 0.0014062 0.0103353 0.0030325 0.0036055 0.0060319 0.008509 T > A A T 0.0017405 0.0060937 0.0027753 0.0049731 0.0013625 0.0051449 0.0032603 0.0050987 T > A C A 0 0.0033265 0.0014546 0.0078634 0.0012155 0.0029254 0.0037785 0.0058558 T > A C C 0.0011786 0.005607 0.0019025 0.0101123 0.0020752 0.0037969 0.0060544 0.0075074 T > A C G 0.0010009 0.0076966 0.0024896 0.0149125 0.003107 0.004439 0.0071564 0.0141123 T > A C T 0.0010563 0.0042866 0.0015635 0.0099675 0.0022963 0.0046869 0.0055776 0.0081023 T > A G A 0 0.0038308 0.000825 0.0049977 0.0031311 0.0032647 0.0036468 0.0069156 T > A G C 0.0040031 0.0057509 0.0006919 0.0053191 0.0024275 0.0028705 0.0038908 0.0058725 T > A G G 0.0007367 0.0071128 0.0016335 0.0068918 0.003584 0.0040722 0.0044361 0.0086161 T > A G T 0.0006885 0.0035647 0.0007714 0.00358 0.0033467 0.0031455 0.0047852 0.0054034 T > A T A 0 0.0029489 0.0006674 0.0062995 0.0020975 0.0035245 0.001789 0.0045921 T > A T C 0.0016816 0.0038294 0.0016767 0.005077 0.0015763 0.0044968 0.0033028 0.0053856 T > A T G 0.0006063 0.0032448 0.0008153 0.0083736 0.0014849 0.0034758 0.0027489 0.0080295 T > A T T 0.0005865 0.0036501 0.0035182 0.0056041 0.0023488 0.0059528 0.0039545 0.0068197 T > C A A 0.0043032 0.0123779 0.0053456 0.0074281 0.0059307 0.0049681 0.0084087 0.0058366 T > C A C 0.0039151 0.0097116 0.0050443 0.0057317 0.0045614 0.0037033 0.0046872 0.0056801 T > C A G 0.0034793 0.0197224 0.0048826 0.0087596 0.0055127 0.0054823 0.0129302 0.0103419 T > C A T 0.0022726 0.0098358 0.0050127 0.0057195 0.0041368 0.0051976 0.0104551 0.0044841 T > C C A 0.0019843 0.0092097 0.0019979 0.0040958 0.0039623 0.0040624 0.0066636 0.0053682 T > C C C 0.0046538 0.0114502 0.0052466 0.0068469 0.0056999 0.0049614 0.0072185 0.0051033 T > C C G 0.0024242 0.0220666 0.0042274 0.0130391 0.0055162 0.0057134 0.0088471 0.0105015 T > C C T 0.0019631 0.0129148 0.0039328 0.0068022 0.0055377 0.0062782 0.0064333 0.0063119 T > C G A 0.0084245 0.0187299 0.0106856 0.0066819 0.0105541 0.0040895 0.0063614 0.0068431 T > C G C 0.0072351 0.0149648 0.0116742 0.0041276 0.0075331 0.0035488 0.0057544 0.0051587 T > C G G 0.0081675 0.019088 0.007555 0.0062124 0.0065655 0.0061082 0.0105821 0.0082351 T > C G T 0.0027813 0.0132836 0.0084494 0.007911 0.0055616 0.0089018 0.0074425 0.0059076 T > C T A 0.00232 0.0096929 0.0033157 0.006331 0.0049397 0.0054976 0.0051958 0.0034565 T > C T C 0.0033433 0.0097908 0.0054282 0.0063694 0.0046408 0.0046156 0.0061161 0.0032024 T > C T G 0.002704 0.0117797 0.0056605 0.0064651 0.0033823 0.0054129 0.004246 0.0043711 T > C T T 0.0029444 0.00896 0.0051954 0.0071857 0.0037035 0.0054088 0.0050798 0.0026365 T > G A A 0.0002595 0.002508 0.0022859 0.0010487 0.0014028 0.0022766 0.002766 0.0020063 T > G A C 0.0013774 0.0035956 0.0038721 0.0024862 0.0006668 0.0022185 0.0032677 0.002079 T > G A G 0.00087 0.0037953 0.0027905 0.0020745 0.0024564 0.0035213 0.004519 0.0035771 T > G A T 0.0029299 0.0034326 0.0090542 0.0021008 0.001523 0.0022227 0.0025711 0.0024628 T > G C A 0.000547 0.0045258 0.0033842 0.0032886 0.0024278 0.0027251 0.0044246 0.0043926 T > G C C 0.0010438 0.0074306 0.0026307 0.0037417 0.0021872 0.0038184 0.0046714 0.0033817 T > G C G 0.0010004 0.0102325 0.0039628 0.0094587 0.0022028 0.0045527 0.0064928 0.0063998 T > G C T 0.0031236 0.0086819 0.0123496 0.0033005 0.0026843 0.0040113 0.0048141 0.0030168 T > G G A 0.0011658 0.0033709 0.0025441 0.0059632 0.0018842 0.0018375 0.0017334 0.0025622 T > G G C 0.0020052 0.0037429 0.0039376 0.0020627 0.0026466 0.0021084 0.0032899 0.0026635 T > G G G 0.001898 0.005848 0.0038156 0.0050707 0.0049083 0.0050319 0.0044534 0.0044921 T > G G T 0.0035029 0.0068018 0.0044141 0.0043791 0.0038 0.003344 0.0040229 0.0030112 T > G T A 0 0.0019468 0.0049872 0.0043885 0.0010459 0.0018732 0.0027206 0.0021444 T > G T C 0.0017265 0.004624 0.0043574 0.002988 0.00285 0.0035284 0.0050522 0.0030884 T > G T G 0.0008683 0.0043617 0.0029477 0.0048767 0.0012387 0.0034131 0.0039386 0.0049239 T > G T T 0.0048424 0.004117 0.0234712 0.0047025 0.0023416 0.0044935 0.004293 0.0030385

For example, in some embodiments the step of assigning may be carried out by categorizing a tumour into a cluster defined by the mean proportion values depicted in Table 1 plus or minus the corresponding standard deviation value depicted in Table 2. In some embodiments the step of assigning may be carried out by categorizing a tumour into a cluster defined by the mean proportion values depicted in Table 1 plus or minus twice the corresponding standard deviation value depicted in Table 2.

In some embodiments, the step of assigning is carried out by categorizing the tumour into a cluster defined by the mean proportion values depicted in Table 1 plus or minus the corresponding 95% confidence (95% CI) interval depicted in Table 3.

TABLE 3 95% Confidence Intervals for the Mean Proportions for Clusters 1 to 8 95% Cl for Clusters Mut 5′ 3′ 1 2 3 4 5 6 7 8 C > A A A 0.0001437 0.000356 0.0008498 0.0042216 0.0008866 0.0004231 0.0011698 0.004503 C > A A C 0.0016929 0.0006238 0.0012144 0.0067309 0.0010746 0.0003571 0.0012781 0.0055508 C > A A G 0 0.0003619 0.0006473 0.0040648 0.0004277 0.0002076 0.0008589 0.0038916 C > A A T 0.0030373 0.0005024 0.0015411 0.0042069 0.0005065 0.0002127 0.0006619 0.0034016 C > A C A 0.0013737 0.0008868 0.0012952 0.0076103 0.0006867 0.0006594 0.0012403 0.0073748 C > A C C 0.0018786 0.0011175 0.0015959 0.0092286 0.0008934 0.0004286 0.0012565 0.0071859 C > A C G 0.0009812 0.0008438 0.0009936 0.0065742 0.0006017 0.0003176 0.0011321 0.005069 C > A C T 0.0113528 0.0020566 0.0036898 0.0071606 0.0004264 0.0004696 0.0011071 0.0060593 C > A G A 0.0007377 0.0005317 0.0009832 0.0055409 0.0008018 0.0003272 0.0010798 0.0044014 C > A G C 0.0022476 0.0006838 0.0009655 0.0058088 0.0008258 0.0003038 0.0013128 0.0058535 C > A G G 0.0010528 0.0005467 0.0008817 0.0078439 0.0002694 0.0002485 0.0007287 0.004503 C > A G T 0.0075875 0.001026 0.0020767 0.0039755 0.0008247 0.0003049 0.0009898 0.0040872 C > A T A 0.0020833 0.0005313 0.0050353 0.0090586 0.0004016 0.0004271 0.0035191 0.0055125 C > A T C 0.0017432 0.0006709 0.0017011 0.0071299 0.0006509 0.0005438 0.0022901 0.0060997 C > A T G 0.0005804 0.000374 0.000972 0.0036078 0.0005252 0.0002497 0.0021072 0.0036928 C > A T T 0.0100513 0.0009779 0.0189334 0.013288 0.0006932 0.0004106 0.0022619 0.0049349 C > G A A 0.0005714 0.0003561 0.0003826 0.0030959 0.0004325 0.0002231 0.0010532 0.0029474 C > G A C 0.0002448 0.0004726 0.000538 0.002215 0.0007042 0.0002574 0.0011851 0.0024232 C > G A G 0.0003663 0.0003929 0.0006146 0.0020331 0.0004716 0.0002706 0.0009293 0.0017399 C > G A T 0 0.0004226 0.0005678 0.0020624 0.0005146 0.0002691 0.0010251 0.0019458 C > G C A 0.0003876 0.0004065 0.000251 0.0029428 0.0005166 0.00033 0.0014239 0.0028583 C > G C C 0.0007641 0.0005636 0.0007551 0.0035193 0.0006497 0.0004027 0.0013769 0.0021314 C > G C G 0.0004413 0.0005349 0.0007533 0.0039477 0.000599 0.000391 0.0012606 0.0026723 C > G C T 0.0003103 0.0004133 0.0007861 0.0029841 0.0009029 0.0003082 0.0013049 0.0023604 C > G G A 0.0003847 0.0003919 0.0005311 0.0033936 0.0003063 0.0001992 0.0007377 0.0018957 C > G G C 0.000423 0.0005688 0.0008795 0.0032822 0.000555 0.0003285 0.0009389 0.0029186 C > G G G 0.0001224 0.0003515 0.0002195 0.0025377 0.0006014 0.0002752 0.0010592 0.0021739 C > G G T 0.0001511 0.0004211 0.0005879 0.002362 0.0010516 0.0002445 0.001202 0.0021791 C > G T A 0 0.0004795 0.0002159 0.0050555 0.0007324 0.0006989 0.0066828 0.0112388 C > G T C 0.0004159 0.0004826 0.000636 0.0042547 0.000524 0.0004805 0.0033823 0.0035426 C > G T G 0 0.0003263 0.0005558 0.0023405 0.0004532 0.0002958 0.0024001 0.0019465 C > G T T 0.000842 0.0007336 0.0006617 0.0043191 0.0009605 0.0007542 0.0074931 0.0171676 C > T A A 0.0036696 0.0013527 0.0007898 0.0041798 0.0021209 0.00046 0.0016767 0.0028545 C > T A C 0.0071607 0.0012211 0.0017137 0.0028963 0.0058216 0.0012773 0.0017091 0.0027339 C > T A G 0.0137265 0.0043681 0.0047272 0.0052975 0.0030915 0.0008329 0.0028941 0.0025958 C > T A T 0.0032041 0.0009137 0.0017902 0.0028438 0.0041657 0.0007267 0.0012295 0.0024813 C > T C A 0.0018484 0.000981 0.0009561 0.0051236 0.0027612 0.0018979 0.0025407 0.0038173 C > T C C 0.0044783 0.0010782 0.0013965 0.0056084 0.0076058 0.0030532 0.0021108 0.0039955 C > T C G 0.0112365 0.0043592 0.0044769 0.0075103 0.0035274 0.0020282 0.0034357 0.0046909 C > T C T 0.0036232 0.0012108 0.0016921 0.0051422 0.0047578 0.0022953 0.0024517 0.0034005 C > T G A 0.006877 0.0018072 0.0014838 0.0037022 0.0025855 0.000521 0.0019961 0.0028085 C > T G C 0.0090539 0.0024365 0.0032692 0.0046119 0.0072714 0.0013533 0.0017535 0.003005 C > T G G 0.0225278 0.006039 0.0104632 0.0036963 0.0037058 0.0008044 0.0031618 0.0030014 C > T G T 0.0047964 0.0018977 0.0024788 0.0034828 0.004797 0.0008919 0.0020041 0.003224 C > T T A 0.0020013 0.0012738 0.0015584 0.004297 0.0025303 0.0026916 0.0099551 0.0089084 C > T T C 0.0056969 0.0010671 0.0024158 0.0037888 0.0064134 0.0037494 0.0047185 0.0043418 C > T T G 0.0154091 0.0026472 0.0189653 0.0048128 0.0020767 0.0030727 0.0050398 0.0060825 C > T T T 0.005689 0.0010187 0.0028113 0.0047619 0.0050331 0.0019356 0.0059117 0.0055715 T > A A A 0.0004359 0.0003588 0.0002301 0.001915 0.0004573 0.0003236 0.0007856 0.0014932 T > A A C 0.001935 0.0005963 0.0004665 0.0025881 0.0005099 0.0002917 0.0006245 0.0015821 T > A A G 0.0006056 0.0003625 0.0003664 0.0036647 0.0005553 0.0002995 0.0009667 0.0026516 T > A A T 0.0009639 0.0005235 0.0007232 0.0017634 0.0002495 0.0004274 0.0005225 0.0015889 T > A C A 0 0.0002858 0.0003791 0.0027883 0.0002226 0.000243 0.0006055 0.0018248 T > A C C 0.0006527 0.0004817 0.0004958 0.0035857 0.00038 0.0003154 0.0009703 0.0023395 T > A C G 0.0005543 0.0006612 0.0006488 0.0052877 0.0005689 0.0003688 0.0011469 0.0043977 T > A C T 0.000585 0.0003682 0.0004074 0.0035343 0.0004205 0.0003894 0.0008939 0.0025249 T > A G A 0 0.0003291 0.000215 0.0017721 0.0005733 0.0002712 0.0005844 0.0021551 T > A G C 0.0022168 0.000494 0.0001803 0.0018861 0.0004445 0.0002385 0.0006235 0.00183 T > A G G 0.0004079 0.000611 0.0004257 0.0024437 0.0006563 0.0003383 0.0007109 0.002685 T > A G T 0.0003813 0.0003062 0.000201 0.0012694 0.0006128 0.0002613 0.0007669 0.0016838 T > A T A 0 0.0002533 0.0001739 0.0022337 0.0003841 0.0002928 0.0002867 0.001431 T > A T C 0.0009312 0.000329 0.000437 0.0018002 0.0002886 0.0003736 0.0005293 0.0016783 T > A T G 0.0003358 0.0002787 0.0002125 0.0029692 0.0002719 0.0002888 0.0004405 0.0025022 T > A T T 0.0003248 0.0003135 0.0009168 0.0019871 0.0004301 0.0004945 0.0006337 0.0021252 T > C A A 0.002383 0.0010633 0.0013931 0.0026339 0.001086 0.0004127 0.0013476 0.0018188 T > C A C 0.0021681 0.0008342 0.0013146 0.0020324 0.0008352 0.0003077 0.0007512 0.00177 T > C A G 0.0019268 0.0016942 0.0012724 0.003106 0.0010094 0.0004555 0.0020722 0.0032228 T > C A T 0.0012585 0.0008449 0.0013063 0.0020281 0.0007575 0.0004318 0.0016755 0.0013973 T > C C A 0.0010989 0.0007911 0.0005207 0.0014523 0.0007255 0.0003375 0.0010679 0.0016728 T > C C C 0.0025772 0.0009836 0.0013673 0.0024278 0.0010437 0.0004122 0.0011568 0.0015903 T > C C G 0.0013425 0.0018956 0.0011017 0.0046235 0.0010101 0.0004747 0.0014178 0.0032725 T > C C T 0.0010871 0.0011094 0.0010249 0.002412 0.001014 0.0005216 0.001031 0.0019669 T > C G A 0.0046653 0.0016089 0.0027847 0.0023693 0.0019326 0.0003398 0.0010195 0.0021325 T > C G C 0.0040066 0.0012855 0.0030423 0.0014636 0.0013794 0.0002948 0.0009222 0.0016076 T > C G G 0.004523 0.0016397 0.0019688 0.0022028 0.0012022 0.0005075 0.0016959 0.0025662 T > C G T 0.0015402 0.0011411 0.0022019 0.0028051 0.0010184 0.0007395 0.0011927 0.0018409 T > C T A 0.0012848 0.0008326 0.0008641 0.0022449 0.0009045 0.0004567 0.0008327 0.0010771 T > C T C 0.0018515 0.0008411 0.0014146 0.0022585 0.0008498 0.0003835 0.0009802 0.0009979 T > C T G 0.0014974 0.0010119 0.0014751 0.0022924 0.0006193 0.0004497 0.0006805 0.0013621 T > C T T 0.0016305 0.0007697 0.0013539 0.002548 0.0006781 0.0004494 0.0008141 0.0008216 T > G A A 0.0001437 0.0002154 0.0005957 0.0003718 0.0002569 0.0001891 0.0004433 0.0006252 T > G A C 0.0007628 0.0003089 0.0010091 0.0008816 0.0001221 0.0001843 0.0005237 0.0006478 T > G A G 0.0004818 0.000326 0.0007272 0.0007356 0.0004498 0.0002925 0.0007242 0.0011147 T > G A T 0.0016225 0.0002949 0.0023595 0.0007449 0.0002789 0.0001847 0.000412 0.0007675 T > G C A 0.0003029 0.0003888 0.0008819 0.0011661 0.0004446 0.0002264 0.0007091 0.0013688 T > G C C 0.000578 0.0006383 0.0006856 0.0013268 0.0004005 0.0003172 0.0007486 0.0010538 T > G C G 0.000554 0.000879 0.0010327 0.0033539 0.0004034 0.0003782 0.0010405 0.0019943 T > G C T 0.0017298 0.0007458 0.0032183 0.0011703 0.0004915 0.0003332 0.0007715 0.0009401 T > G G A 0.0006456 0.0002896 0.000663 0.0021145 0.000345 0.0001527 0.0002778 0.0007984 T > G G C 0.0011105 0.0003215 0.0010261 0.0007314 0.0004846 0.0001752 0.0005272 0.00083 T > G G G 0.0010511 0.0005024 0.0009944 0.001798 0.0008988 0.000418 0.0007137 0.0013998 T > G G T 0.0019398 0.0005843 0.0011503 0.0015527 0.0006958 0.0002778 0.0006447 0.0009384 T > G T A 0 0.0001672 0.0012997 0.0015561 0.0001915 0.0001556 0.000436 0.0006682 T > G T C 0.0009561 0.0003972 0.0011356 0.0010595 0.0005219 0.0002931 0.0008097 0.0009624 T > G T G 0.0004809 0.0003747 0.0007682 0.0017292 0.0002268 0.0002836 0.0006312 0.0015344 T > G T T 0.0026816 0.0003537 0.0061166 0.0016674 0.0004288 0.0003733 0.000688 0.0009469

In some embodiments involving ranges, the categorizing is based on highest cosine similarity to the mean proportion values for the ranges established, as described above, using mean proportion values depicted in Table 1 and the standard deviation value depicted in Table 2; or the mean proportion values depicted in Table 1 and the 95% CI values depicted in Table 3.

In some of these embodiments, a minimum of 0.75 cosine similarity may be employed as a threshold value for the assignment. In another embodiment, the threshold may be 0.7. In another embodiment, the threshold may be 0.8. In another embodiment, the threshold may be 0.9. In another embodiment, the threshold may be 0.95.

In another embodiment, the categorizing is by clustering. For example, in one embodiment, the categorizing may be based on based on distance. For example Levenshtein's distance may be used for the categorizing. For example, Euler's distance may be used for the categorizing. Other distance metrics may also be used, according to requirements.

By “Mutation”, as used herein, will be understood as any sequence difference relative to reference sequence. Reference sequences may be selected according to a patient demographic. Reference sequences may be selected according to a sample type. By “early mutation”, as used herein, is meant a mutation that occurs relatively early in tumourigenesis. Early mutations are, in some cases, known to influence tumour biology and/or the course of subsequent tumourigenesis. By “germline mutation”, as used herein, is meant a subset of early mutations that are inherited, therefore constitutional in the subject. The subject may also be “mosaic” for some early mutations, meaning that only some somatic cells possess the mutation. A mosaic mutation may be present the entirety of cells of one or more lineage. By “driver mutation”, as used herein, is meant a mutation that has contributed to tumourigenesis, and/or a mutation responsible for an aspect of current tumour biology. Such mutations could encompass mutations in an oncogene or a tumour suppressor.

The term “hypermutant”, as used herein will be understood as any tumour bearing the requisite number of mutations per megabase (mut/Mb). A hypermutant tumour may comprise at least 3 mut/Mb, at least 4 mut/Mb, at least 5 mut/Mb, at least 6 mut/Mb, at least 7 mut/Mb, at least 8 mut/Mb, at least 9 mut/Mb, or at least 10 mut/Mb. It will be understood that the term “hypermutant” also encompasses “ultra-hypermutant”, which tumours have a higher mutational burden. An ultra-hypermutant tumour may comprise at least 50 mut/Mb, at least 60 mut/Mb, at least 70 mut/Mb, at least 80 mut/Mb, at least 90 mut/Mb, or at least 100 mut/Mb. It will be further understood that the thresholds disclosed herein may be specific to the platform selected for determining mutations, and/o to the regions of DNA sequenced. These thresholds could be readily adapted and/or cognate thresholds could be established for other platforms.

By “deficient”, as used herein with respect to a cellular state or pathway, will be understood as in comparison to normal tissue (e.g. healthy tissue), for example a healthy tissues-matched sample.

In one embodiment, the at least one tumour characteristic comprises timing of a mutagenic event in the development of the tumour. In one embodiment, the at least on tumour characteristic comprises an early mutation. In one embodiment, the at least one tumour characteristic comprises a germline mutation. In one embodiment, the at least one tumour characteristic comprises exposure to a mutagen. In one embodiment, the at least one tumour characteristic is a relative order of at least two tumourigenic events. In one embodiment, the at least one tumour characteristic comprises tissue origin. Information about tumour origin may help to identify metastatic tumours.

In one embodiment, the at least one tumour characteristic is further determined based on the presence of one or more driver mutation as defined in Table 6. In one embodiment, the one or more driver mutation comprise one or more of the mutations labelled ‘New’ in Table 6.

In one embodiment, the hypermutant tumour has mutation frequency of at least 5 mutation per megabase, and the tumour is a pediatric tumour. In one embodiment, the hypermutant tumour has a mutation frequency of at least 9 mutation per megabase. In one embodiment, the hypermutant tumour has a mutation frequency of at least 9.9 mutations per megabase, and the tumour is from an adult. In one embodiment, the hypermutant tumour is an ultra-hypermutant tumour having a mutation frequency of at least 100 mutations per megabase.

In one embodiment, wherein the nucleic acid comprises DNA.

In one embodiment, the nucleic acid comprises RNA.

In one embodiment, the sequencing is targeted sequencing. In one embodiment, the targeted sequencing is as defined herein the Examples.

In one embodiment, the sequencing is whole exome sequencing.

In one embodiment, the sequencing is whole genome sequencing.

Cluster 1

In one embodiment, cluster 1 is indicative of a hypermutant tumour with microsatellite stability. This hypermutant tumour indicated by cluster 1 may be ultra-hypermutant. In one embodiment, cluster 1 is indicative of an MMR gene mutation. In one embodiment, the MMR gene mutation is a germline mutation. In one embodiment, cluster 1 is indicative of a POLE gene mutation. In one embodiment, the POLE gene mutation indicated by cluster 1 is secondary to an MMR gene mutation.

Cluster 2

In one embodiment, cluster 2 is indicative of a hypermutant tumour with microsatellite instability. In one embodiment, cluster 2 is indicative of an MMR gene mutation. In one embodiment, the MMR gene mutation indicated by cluster 2 is an early MMR gene mutation.

Cluster 3

In one embodiment, cluster 3 is indicative of a hypermutant tumour with microsatellite stability. This hypermutant tumour indicated by cluster 3 may be ultra-hypermutant. The method of any one of claims 1 to 17, wherein cluster 3 is indicative of a POLE gene mutation. In one embodiment, the POLE mutation indicated by cluster 3 is an early POLE gene mutation. In one embodiment, cluster 3 is indicative of MMR gene mutation. In one embodiment, the MMR gene mutation indicated by cluster 3 is secondary to a POLE gene mutation.

Cluster 4

In one embodiment, cluster 4 is indicative of exposure to a mutagen in tobacco smoke. In one embodiment, cluster 4 is indicative of lung cancer. In one embodiment, the lung cancer is metastatic lung cancer. In one embodiment, this metastatic lung cancer may have been previously misdiagnosed.

Cluster 5

In one embodiment, cluster 5 is indicative of exposure to an alkylating agent. In one embodiment, cluster 5 is indicative of prior treatment with an alkylating agent. In one embodiment, cluster 5 is indicative of tumour resistance to alkylating agents.

Cluster 6

In one embodiment, cluster 6 is indicative of exposure to UV light. In one embodiment, cluster 6 is indicative of skin cancer. In one embodiment, the skin cancer is metastatic skin cancer. This metastatic skin cancer may have been previously misdiagnosed. In one embodiment, cluster 6 is indicative of a sarcoma induced by UV light exposure. In one embodiment, the sarcoma is metastatic. This metastatic sarcoma may have been previously misdiagnosed.

Cluster 7

In one embodiment, cluster 7 is indicative of deficient APOBEC cytidine deamination.

Cluster 8

In one embodiment, cluster 8 is indicative of exposure to a mutagen in tobacco smoke. In one embodiment, cluster 8 is indicative of lung cancer. In one embodiment, the lung cancer is metastatic lung cancer. In one embodiment, this metastatic lung cancer may have been previously misdiagnosed.

Medical Treatments and Uses

In one aspect, the above-described methods permit treatment and/or clinical intervention to be selected or applied based on the determined at least one tumour characteristic. Any suitable treatment and/or clinical intervention may be used based on the tumour characteristic(s) associated with the cluster(s) to which the tumour is assigned. For example, a suitable therapeutic agent could be selected based on the tumour characteristic.

In one embodiment, the method further comprises selecting a treatment for the patient from whom the tumour was obtained based on the at least one tumour characteristic determined with one of the above-described methods.

In one embodiment, the method further comprises treatment of a patient from whom the tumour was obtained based on the at least one tumour characteristic determined with one of the above-described methods.

In one embodiment, the tumour is matched to any one of clusters 1 to 3, and the treatment comprises immunotherapy. In one embodiment, the tumour is matched to any one of clusters 1 to 3, and the treatment comprises an immune checkpoint inhibitor.

In one embodiment, the tumour is matched to cluster 4 or 8 and the treatment comprises a lung cancer treatment.

In one embodiment, the tumour is matched to cluster 5 and the treatment does not comprise an alkylating agent. A treatment may be selected, in this case, for tumours known to be resistant to alkylating agents.

In one embodiment, the tumour is matched to cluster 5 and the treatment is skin cancer treatment. In one embodiment, the tumour is matched to cluster 5 and the treatment is treatment for sarcoma induced by UV light.

In one embodiment, the treatment is based on reclassification of the tumour based on the determined at least one tumour characteristic. Applying the above-described method may reveal misclassification or previously unknown details regarding e.g. tumour origin or tissue type, leading to reclassification or re-diagnosis. Treatment may be selected or applied on this basis.

In one embodiment, there is provided a use of a therapeutic agent, selected based on the at least one tumour characteristic identified by one of the above-described method, for treatment a subject from whom the sample was obtained.

In one embodiment, there is provided a use of a therapeutic agent, selected based on the at least one tumour characteristic identified by one of the above-described method, for preparation of a medicament for treatment of a subject from whom the sample was obtained.

In one embodiment, there is provided a therapeutic agent, selected based on the at least one tumour characteristic identified by one of the above-described method, for use in treatment of a subject from whom the sample was obtained.

By “therapeutic agent” is mean any small molecule or biologic suitable for treatment of the tumour.

In one embodiment, the method further comprises selecting a clinical intervention for the patient from whom the tumour was obtained based on the at least one tumour characteristic.

In one embodiment, the clinical intervention is based on reclassification of the tumour based on the at least one tumour characteristic.

In one embodiment, the tumour is matched to any one of clusters 1 to 3, and the clinical intervention comprises clinical surveillance for additional tumours caused by a germline mutation.

In one embodiment, the tumour is matched to any one of clusters 1 to 3, and the clinical intervention comprises genetic counseling or screening for a germline mutation. This may be extended to encompass methods comprising screening or counseling for family members who may also carry the germline mutation.

In one embodiment, there is provided a use of a tumour treatment for treatment of a patient comprising a tumour, wherein the tumour has been profiled according to the method of any one of the above-described methods, and wherein the tumour treatment has been determined based on the assigning of the tumour to a cluster.

In one embodiment, there is provided a tumour treatment for use in treatment of a patient comprising a tumour, wherein the tumour has been profiled according to the method of any one of the above-described methods, and wherein the tumour treatment has been determined based on the assigning of the tumour to a cluster.

In one embodiment, the tumour has been assigned to any one of clusters 1 to 3, and the tumour treatment comprises immunotherapy.

In one embodiment, the tumour has been assigned to any one of clusters 1 to 3, and the tumour treatment comprises an immune checkpoint inhibitor.

In one embodiment, the tumour has been assigned to cluster 5 and the tumour treatment is free of alkylating agents.

Devices and Automation

The above described method could be computer-implemented in some embodiments. A computational device could be configured to perform the above-describe methods. The device could output the assignment for a given tumour to print-out or display. The device could record or transmit the assignment. The device could also output, display, record, and/or transmit a pertinent confidence rating for the result, based on the step of assigning. The device may be configured to process the sample, e.g. to carry out nucleic acid extraction on the tumour sample. The device could be configured to carry out the sequencing. The device could comprise a processed programmed to identify mutations, classify mutations, and carry out the assigning.

The methods or devices described herein may be particularly useful in clinical settings in which extensive histological, pathological, cytological, and/or genetic testing capabilities are limited or otherwise unavailable.

EXAMPLES

Introduction

Mutations in cancer genes can be inherited, arise spontaneously in pre-malignant cells or be acquired over time during tumour evolution (Stratton et al., 2009). It is often difficult to determine, from the sequencing of the diagnostic specimen alone, which mutations arose first or whether their order is important. The ultimate aggregate mutation count in a tumour, termed mutation burden, is highly tissue type dependent. For example, the mutation burden of Ewing sarcoma is two orders of magnitude less than that of adult melanoma (Brohl et al., 2014). Although most tumours are driven by a select few major genetic and epigenetic events and have similar mutation burdens, there is emerging evidence that outliers with much higher mutation burdens (hypermutation) exist for many cancer types. Systematic retrospective sequencing efforts such as those of The Cancer Genome Atlas (TOGA) and the International Cancer Genome Consortium (ICGC) have confirmed frequent hypermutation in melanoma (Cancer Genome Atlas, 2015), lung (Govindan et al., 2012), bladder cancer (Cancer Genome Atlas Research, 2014) and uncovered rare cases of hypermutation in other cancers. However, these studies have used different thresholds as there is no agreed-upon definition of “hypermutation”. Larger unbiased cohorts are needed to define the cut-offs for, identify the extent of, as well as the causes of hypermutation across human cancer.

Determining the frequency and causes of hypermutation, as well the temporal order in which it arises, is of immediate clinical importance. First, there is an urgent need to define driver mutations in cancer genomes. This is especially difficult in hypermutant cancers due to the sheer abundance of passenger variants that can obscure true drivers. Second, understanding early drivers of hypermutation may be useful for predicting the cancer's evolutionary trajectory and accumulation of additional mutations. Finally, hypermutation is strongly correlated with response to immune checkpoint inhibitors, which can lead to durable remissions in some patients (Bouffet et al., 2016; Johanns et al., 2016; Le et al., 2015; Rizvi et al., 2015; Santin et al., 2016; Van Allen et al., 2015).

Hypermutation can be caused by environmental factors (extrinsic exposures). UV light is the primary cause of the accumulation of high numbers of mutations in malignant melanoma (Pfeifer et al., 2005; Sage, 1993). Similarly, the >60 carcinogens in tobacco smoke are the primary cause of hypermutation in lung, larynx and many other tumours, due to direct mutagen exposure (Pleasance et al., 2010). Recently, several intrinsic sources of hypermutation have been described. Dysregulation of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family members, a group of cytidine deaminases, has been shown to result in increased levels of C to T transitions in a wide range of tumour types including breast, bladder and cervical cancer (Roberts et al., 2013). Defective DNA replication repair by mutations that compromise either proofreading, performed by the major replicative enzymes POLE and POLD1, or DNA mismatch repair, are associated with hypermutation in colorectal, endometrial and other cancers (Kandoth et al., 2013; Network, 2012). DNA replication repair mutations are also found in cancer predisposition syndromes, such as constitutional or biallelic mismatch repair deficiency (CMMRD), Lynch, and polymerase proofreading-associated polyposis (PPAP). While data gathered by the international bMMRD (biallelic mismatch repair deficiency) consortium revealed that all malignant CMMRD cancers are hypermutant (Bouffet et al., 2016), it is not known if the same is true in Lynch or PPAP. Furthermore, replication repair defects can lead to acquired resistance to common genotoxic therapies such as alkylating agents (van Thuijl et al., 2015). From the tumour sequence alone, it is currently impossible to define the sequence of events or the order of mutagen exposure leading to hypermutation.

Mutagenic processes leave imprints on the genome in the form of mutations arising in a specific nucleotide context that, when considered together, form a unique signature. This is especially true for hypermutant cancers whose vast numbers of non-random mutations form a signature that is deeply engraved on the cancer genome. The first taxonomic classification of signatures unveiled >20 signatures in 30 cancer types (Alexandrov et al., 2013). From this publication and subsequent work showing at least 30 signatures (Morganella et al., 2016; Nik-Zainal et al., 2016) with the common hypermutation-associated signatures have now become well characterized. However, the driving forces of rarer hypermutation-associated signatures, seen in fewer tumours, are mostly unknown.

Here, we examined 78,452 adult and 2,885 childhood cancers for hypermutation. Targeted regions of the genome were deeply sequenced using a validated cancer gene panel platform (Frampton et al., 2013). We analyzed the range and frequency of hypermutation between and within cancer types, measured the contribution of intrinsic and extrinsic mutators and used mutation signatures (also termed “clusters”) to accurately predict past mutagen exposure. We also used exome sequencing of patients with CMMRD—whose mutations were acquired in an established order (Shlien et al., 2015)—as well as cancers whose hypermutation was treatment-induced to model mutation dynamics in the tumours. The data here have important implications for our understanding of how mutagenic forces govern tumour development and progression.

Methods

Patient and Sample Collection for Exome Sequencing

A cohort of germline replication-repair deficient patients with known clinical history was collected as described previously (Shlien et al., 2015). In brief, patients were registered as a part of the International Biallelic Mismatch Repair Consortium, which includes multiple centers worldwide. Following Institutional Research Ethics Board approval, all data were centralized in the Division of Haematology/Oncology at The Hospital for Sick Children (SickKids). Consent forms were obtained from the parents or guardians, or from the patients, where applicable. Family history, demographic and clinical data were obtained from the responsible physician and/or genetic counselor at the corresponding centers. Tumour and blood samples were collected from the SickKids tumour bank. The diagnosis of a replication repair deficiency-related cancer predisposition syndrome was made when a germline biallelic mutation in any of the four MMR genes (MLH1, MSH2, MSH6, PMS2) or a driver mutation in POLE, was confirmed by sequencing in a clinically approved laboratory. The sequencing of temozolomide-treated tumours was previously described (van Thuijl et al., 2015).

FoundationOne Panel Sequencing

FoundationOne Panel sequencing was performed for 81,337 tumours as previously described (Frampton et al., 2013). In brief, exonic hybridization capture of 315 cancer-related genes was applied to a minimum of 50 ng of DNA extracted from formalin-fixed paraffin-embedded clinical cancer specimens. Pathologic diagnosis of each case was confirmed by review of hematoxylin and eosin (H&E) stained slides and samples were excluded if found to contain <20% tumour cells. Libraries were sequenced to high uniform median coverage (>500×) and assessed for base substitutions, copy number alterations, and gene fusions/rearrangements. For the purposes of the findings described in this study pediatric was defined as <25 years of age.

Exome Sequencing

High-throughput sequencing, read mapping and identification of mutations was performed at the Center for Applied Genomics at the Hospital for Sick Children, as previously described (Shlien et al., 2015). Briefly, tumour and matched blood derived DNA were run using Agilent's exome enrichment kit (Sure Select V4/V5; with >50% of baits above 25× coverage), on an Illumina HiSeq2500. Base calls and intensities from the Illumina HiSeq 2500 were processed into FASTQ files using CASAVA and/or HAS. The paired-end FASTQ files were aligned to UCSC's hg19 GRCh37 with BWA. Aligned reads were realigned for known insertion/deletion events using SRMA and/or GATK. Base quality scores were recalibrated using the Genome Analysis Toolkit26 (v1.1-28). Somatic substitutions were identified using MuTect (v1.1.4). Mutations were then filtered against common single-nucleotide polymorphisms (SNPs) found in dbSNP (v132), the 1000 Genomes Project (February 2012), a 69-sample Complete Genomics data set, and the Exome Sequencing Project (v6500) and the ExAc database.

Whole Genome Sequencing

Whole genome sequencing was performed at The Centre for Applied Genomics on an Illumina HiSeq 2500 or Illumina HiseqX at mean coverage >=30. Read alignment and variant calling/filtering were performed as described above for exome sequencing.

KiCS Panel Sequencing

The SickKids Cancer Sequencing (KiCS) gene panel utilizes the Agilent Sure Select capture kit technology, targeting 15,000 exons across 880 genes. Enriched libraries were prepared from both tumour DNA and matched normal (blood or skin) and sequenced on Illumina HiSeq2500 sequencers running in rapid mode producing paired end 100 base reads. Reads were aligned with BWA-MEM according to GATK best practices with coverage metrics meeting greater than 700× mean coverage, with >=98.5% of bases above 50×, >=95% of bases above 200× coverage, and >=75% of bases above 500× coverage. Substitution mutations were called using MuTect, with variants called above 50× coverage in tumour and normal. We achieved >95% sensitivity and specificity for variants above 5% allele frequency.

Determination of Hypermutation Threshold

In order to determine a threshold of hypermutation in human cancer, a segmented linear regression analysis or “broken-stick analysis” was performed on the mutation burdens from the pediatric (n=2,885) and adult (n=78,452) FoundationOne panel cohorts. Briefly, using the R package Segmented (Muggeo et al., 2003), an iterative process was used to determine segment breakpoints at which a statistically significant change in the slope of adjacent regression lines occurred. For the pediatric cohort, the first such breakpoint at which a statistically significant change occurred, accompanied by a visually observed uptick in the slope of the regression line, was at 9.91 mut/Mb. For the adult cohort a corresponding change occurred at 9 mut/Mb. This threshold was rounded up to 10 mut/Mb to account for statistical uncertainty and the purposes of simplicity.

Microsatellite Instability Analysis

To determine MSI status, 114 intronic homopolymer repeat loci with adequate coverage on the FoundationOne panel are analyzed for length variability and compiled into an overall MSI score via principal components analysis (PCA). Amongst the 1,897 microsatellites, the 114 that maximized variability between samples were chosen. Each chosen locus was intronic and had hg19 reference repeat length of 10-20 bp. This range of repeat lengths was selected such that the microsatellites are long enough to produce a high rate of DNA polymerase slippage, while short enough such that they are well within the 49 bp read length of NGS to facilitate alignment to the human reference genome. A detailed description can be found at Chalmer, Donelly et al., 2017, Genome Research in press.

Detection of POLE and POLD1 Driver Mutations

To identify polymerase mutations associated with hypermutation we built a model based on the following criteria: 1) Tumours harboring the variant must be hypermutant at a conservative cut-off of 50 mutations/mb. This number was selected since most POLE mutant tumours typically exceed 100 mut/mb (Shinbrot et al. 2014). 2) Variants found in hypermutant tumours must not co-occur with an existing known driver mutations in the same tumour. 3) Variants that were found both in hypermutant and lowly mutated tumours (<10 mut/mb) were excluded. 4) Variants must occur in a minimum of 2 hypermutant tumours. Variant allele fraction and tumour purity were also considered, as previously described (Frampton et al., 2013).

POLE Exonuclease Excision Rate Assay

Excision rate constants were measured as described (Zahurancik et al., NAR 2014). Briefly, a pre-incubated solution of Pol e (100 nM) and 5′-32P-labeled DNA substrate (20 nM) was rapidly mixed with Mg2+(8 mM) in reaction buffer at 37° C. After various incubation times, the reaction was quenched with the addition of EDTA. The excision rate constants for Polc wild type and L424V were measured using a rapid chemical quench-flow apparatus. Product concentration was plotted versus time and fit to a single-exponential equation, [product]=Aexp(−k_(exo)t), to yield the excision rate constant, k_(exo).

Unsupervised Clustering and Signature Analysis

A cohort of hypermutant samples was selected for hierarchal clustering and signature analysis by the following criteria.

Exclusion for all:

-   -   1. <50 exonic mutations detected in the FoundationOne panel.

Inclusion for Adults (>25 Years):

-   -   1. Any colon or uterine with MSI-H     -   2. Top 100 most-mutated lung cancers     -   3. Top 100 most-mutated skin cancers     -   4. For all other tumours, >50 Mut/Mb and/or >2 standard         deviations above mean for that tumour type (provided there         were >50 tumours of that type)

Inclusion for Children:

-   -   1. Any tumour with >50 exonic mutations.

This yielded a total of 1,521 tumours (1,491 adult and 30 pediatric) for clustering analysis.

The proportion of mutations corresponding to each of 96 trinucleotide contexts was determined for each of the 1,521 samples selected above using the pyrimidine-converted single base substitution and the corresponding tri-nucleotide sequence context (i.e., reference base at mutation position and its 5′ and 3′ neighbors). Unsupervised hierarchical clustering of the hypermutant cohort by trinucleotide context was performed using the diana clustering method.

The R package DeConstructSigs (Rosenthal et al., 2016) was used to determine the proportion of COSMIC signatures as defined by Alexandrov et al (http://cancer.sangerac.uk/cancergenome/assets/signatures_probabilities.txt) (Alexandrov et al., 2013).

Validation of Signature 7 in Sarcomas Using the TCGA Database

Somatic substitution calls (MuTect2) were obtained from 103 TCGA adult sarcoma exome samples, with a minimum of 50 substitutions. These samples were analysed using deconstructSigs as described above. Five tumours were found to be hypermutant (>10 Mut/Mb) and have significant contributions from Signature 7 (>0.5). Examination of the pathology reports of these tumours revealed all 5 to be located superficially (subcutaneous and/or extending into the dermis).

Subclonal Analysis

Tumour subclones (early and late) were determined using the R package SciClone.8 Variant allele fraction and percentage of alternate reads were used to determine the order of mutational events. Mutations with variant allele fraction greater than 0.45 were excluded from analysis to filter out germline mutations and somatic mutations in regions of copy number gain. For the determination of early and late mutations, clusters were limited to 6 (Miller et al., 2014). Subclonal mutational signatures were then determined using deconstructSigs as described above.

Clinical Genetic Information Collection for Ultra-Hypermutant Pediatric Cancers

Patients with FoundationOne panel results and who were concurrently enrolled in the International Biallelic Mismatch Repair Deficiency Consortium underwent germline sequencing services in accordance with the Clinical Laboratory Improvement Amendments (CLIA) program standards, following a rigorous consent process and genetic counseling consultation. Clinical diagnoses of a replication repair deficiency associated syndrome were made based on the presence of inherited predicted pathogenic mutations in MMR and polymerase genes. Physicians involved in patient care were notified of the diagnosis.

Results

Hypermutation is More Common than Previously Appreciated in Both Childhood and Adult Cancer

We first wanted to define a minimal threshold for hypermutation—to create a common definition that could ultimately be used in a clinical setting. We sequenced a cohort of 35 tumour samples with low, medium and high number of substitutions by exome, genome and two separate targeted panel sequencing consisting of 315 and 884 genes covering 1.1 and 3.25 Mb, respectively (Table 4). We achieved excellent concordance in mutation burden between sequencing modalities when comparing either panel to the genome, exome or both (R2=0.94, FIG. 6 ). After carefully considering sequencing depth, the mutations' allele fractions, and the total footprint of each method, we found that every hypermutated cancer (>10 Mut/Mb) was successfully called by all sequencing methods.

Having validated panel-based hypermutation testing, we examined the mutation burden in 2,885 pediatric tumours. Mutation frequency ranged from 0-864 Mut/Mb (FIG. 1A), with a mean and median of 6.78 Mut/Mb and 2.50 Mut/Mb, respectively. Using segmented linear regression analysis, we calculated 9.91 and 9.0 Mut/Mb as appropriate thresholds for hypermutation in childhood and adult cancers (FIG. 7 and Methods). For consistency, we use 10 Mut/Mb to define hypermutation in both cohorts. We also note that this coincides with the median mutation burden of patients previously reported to respond to checkpoint inhibition (Bouffet et al., 2016; Diaz and Le, 2015; Johanns et al., 2016; Le et al., 2015; Rizvi et al., 2015; Santin et al., 2016; Snyder et al., 2014; Van Allen et al., 2015).

While childhood cancer genomes are typically thought of as ‘quiet’, we found 160 tumours with >10 Mut/Mb (5.5%). Hypermutation was observed in childhood cancer types not typically associated with elevated numbers of mutations, including sarcomas, germ cell tumours, nephroblastomas and neuroblastomas. Importantly, across the whole cohort, hypermutant cancers were enriched for defects in mismatch repair pathway genes POLE and POLD1, responsible for synthesis of the leading and lagging strand (P=<2.2×10-16, FIG. 1B). Pediatric tumours with >100 Mut/Mb—defined as ultra-hypermutated—were universally replication repair deficient. These were limited to malignant gliomas, colorectal cancers and leukemias/lymphomas—that is, the three tumour types observed in CMMRD syndrome.

Replication Repair Deficiency Drives a Mutator Phenotype in Many Tumour Types

Functioning DNA replication repair is required for every actively dividing cell in all tissue types. Yet, thus far, replication repair deficient hypermutation has been mainly observed in gliomas, gastrointestinal tract cancers and endometrial cancers.

We expanded our analysis of hypermutation to 78,452 adult cancers sequenced on the same targeted gene panel. Hypermutation and ultra-hypermutation were observed in 17% and 0.6%, respectively, of cases across a wide variety of tissues (FIGS. 1C and D). A close examination of these cancers revealed enrichment of replication repair mutations and microsatellite instability in 81 tumour types (FIG. 8 ). These include previously underreported hypermutant prostate, cervical and neuroendocrine tumours (87, 84 and 278 cancers).

A striking inverse association was observed between microsatellite instability (MSI), a marker of mismatch repair deficiency, and mutation burden. High levels of microsatellite instability (MSI-H) was mostly restricted to tumours in the 10-100 Mut/Mb range, whereas tumours with >100 Mut/Mb were microsatellite stable and enriched for replicative polymerase mutations. Thus, the loss of mismatch repair ability alone is mutagenic up to a point, while the additional loss of polymerase proofreading, causing total replication repair deficiency, leads to an ultra-hypermutated state with microsatellite stability. These data suggest that tumours with complete replication repair deficiency have elevated mutation burdens, acquired with different temporal dynamics (FIG. 1E).

Detection and Validation of Cancer Drivers Using an “In Vivo Human Mutagenesis Screen”

Rare variants, whether found in the germline or acquired by a tumour, can be difficult to interpret on their own. In many cases, there is no functional assay available for these variants, and most are therefore typically classified as “variants of unknown significance” or “likely passengers” (depending if found in non-neoplastic or cancer material). A handful of missense mutations in the exonuclease domain of POLE have been established as driver variants—that is, they are known to cause hypermutation in human tumours and are functionally validated as drivers in cell line and animal models (Albertson et al., 2009; Daee et al., 2010; Kane and Shcherbakova, 2014). However, determining the pathogenicity of novel mutations in POLE and POLD1 is a challenge due to the large size of these genes and the frequency at which they can be mutated without functional impact. Indeed, in our cohort we observed 2,150 POLE and 1,123 POLD1 distinct variants, many of which encode changes at novel positions in the protein (Methods). As expected, among childhood cancers, most POLE and POLD1 mutations were not associated with hypermutation (FIG. 9 ).

We reasoned that we could determine which variants are in fact true drivers by leveraging the large size of this cohort. A mutation was classified as a clear driver if it was found in a hypermutated tumour and was not observed in tumours with a low mutation burden (Methods). Using this approach—which can be thought of as an “in vivo human mutagenesis screen”—we re-discovered every known POLE and POLD1 driver (that is, every previously published and established driver mutation was correctly picked up by our screen). We then found 11 new driver mutations that are all consistently associated with hypermutation (7 new drivers in POLE and 4 in POLD1; Table 5). For POLD1, the lagging strand polymerase, driver mutations are less well characterized. POLD1 R689W was the most frequent driver in this gene. This variant has been shown to be a very strong mutator in yeast (Daee et al., 2010). To our knowledge this is the first series of primary tumours that validates this variant as a mutator in humans. The absence of hypermutation was also interesting—tumours with exonuclease mutations at highly conserved motifs of POLD1 (ExoI,II,III) were not consistently hypermutant. These variants may be so detrimental to the cell that it requires additional suppressor mutations to reduce the mutation burden (Herr et al., 2011). Just as importantly, through this analysis of POLE and POLD1 mutations, we determined that many mutations are mere passengers, even in tumours with multiple polymerase mutations (FIG. 2A). POLE R446Q is one such false positive—observed in a large group of non-hypermutated cancers. Confirming our classification as non-pathogenic, we also found this variant in non-affected individuals (>1/2000 individuals in ExAC (Lek et al., 2016)). In both POLE and POLD1, driver mutations were uncovered outside the exonuclease domain (FIG. 2B) suggesting that other domains are responsible for hypermutation.

These data allowed us to compare the impact of differing missense substitutions at the same residue. For example, we found that POLE V411 is associated with an extremely high mutation burden but only when valine is mutated to leucine (we labeled these residues as “invariable” as only a single possible amino acid change was found; FIG. 2C). Residues S461, Y458 and E978 in POLE seem to be insensitive to change, with all amino acid substitutions associated with hypermutation. In contrast, at other residues, the magnitude of mutation burden varies depending on the specific amino acid change. Leucine 424 is one such “sensitive” residue—it is associated with a strong mutator phenotype when replaced with a proline or isoleucine, but is associated with lower mutation burden when mutated to a valine or phenylalanine (p=0.03). To validate this finding, we measured the excision rate constants for wild type POLE, and the L424V and L424I mutations using a rapid chemical quench-flow apparatus ((Zahurancik et al., 2014); Methods). L424I had a 5.7-fold stronger excision effect than L424V, confirming the dramatic difference in mutation burden seen in the primary tumours (FIG. 10 ). In contrast, every D275 mutation mutates an essential active site residue, which coordinates a metal ion required for catalysis, and yet we see a 10-fold difference in mutation burden (between D275G, D275A and D275V). The difference between sensitive and insensitive amino acids can be related to the structure of the exonuclease and physical/biochemical interactions with the mismatches in the DNA.

Using this screen to triage functional variants, we mapped the landscape of driver somatic mutation in POLE and POLD1. These observations are key in the precise definition of real drivers in POLE and POLD1 cancers and can solve some issues raised by recent sequencing efforts, which uncovered POLE and POLD1 germline variants of unknown significance in children with cancer (Zhang et al., 2016).

Finally, although most POLE and POLD1 driver mutations were restricted to specific tumour types with no previously apparent signature (FIG. 2D), driver mutations were also observed in melanoma and lung cancer, both of which are malignancies with well-described extrinsic causes of hypermutation and a distinct signature. These data suggest that hypermutation can arise from a variety of sources in a single tumour histotype.

Previous Mutagen Exposure can be Inferred from the Cancer Genome.

To study the context and footprints of hypermutation across all cancers, we studied 217,086 mutations in 1,521 hypermutant tumours representing to our knowledge the largest collection of hypermutated cancers to be considered together (Table 6 and Methods). We classified every mutation, whether coding or not, by its nucleotide context (i.e., the bases immediately preceding and following it, forming a trinucleotide). Then, using the proportion of the 96 possible trinucleotides, we performed unsupervised clustering of all 1,521 tumours. Taking the whole map into view, several known clusters emerged, including replication repair dominated tumours (shown on the left in FIG. 3A), skin cancers known to have a distinctive UV-associated signature, and two tobacco smoking clusters (shown on the right in FIG. 3A).

Strikingly, within the replication repair associated tumours, one sees at least three sharply delineated sub-clusters (labeled C1, C2 and C3 in FIG. 3B). The largest group, C2, (n=523), is characterized by high microsatellite instability in a wide range of cancers (43 tumour types with >2 examples each). The middle-sized cluster, C3, which includes colorectal, uterine and seven other tumour types, is made up of cancers that are almost completely microsatellite stable and POLE mutated. The last DNA replication repair cluster, C1, includes many microsatellite stable brain cancers with mutated POLE. C1 is unique since it harbors a higher mutation burden (380.8 vs 277.5 Mut/Mb for C3 (p=0.017) and 80.4 Mut/Mb for C2 (p=8.9E-5) and also includes many children. Taken together, these results expand the spectrum of cancers associated with DNA replication repair and define three new subgroups—based on trinucleotide mutation context alone—with dramatic differences in microsatellite stability, polymerase deficiency, and age.

The non-replication repair deficient cancers comprised the remaining clusters (labeled C4 to C8 in FIG. 3A). Viewing the overall landscape, these could be seen as roughly divided by already known mutagenic signatures (Alexandrov et al., 2013): tobacco smoke (cluster C4 and C8), alkylating agents (C5), ultraviolet light (C6) and the APOBEC cytidine deamination signature (C7). These four mutagens “anchor” their clusters, which contain the expected cancer types (e.g. cluster C6 has a strong core of UV-associated skin cancers). There were additional smaller clusters of cancers that grouped together (unlabeled in FIG. 3A). Six tumours, including three urothelial and one hepatocellular carcinoma shared a spectrum suggestive of exposure to aristocholic acid (Poon et al., 2013). However, it is clear from our results that the major mutagens act more broadly than previously appreciated and that the canonical mutagen-tumour type relationships do not always hold, or are not exclusive.

Lung cancer is one such example (FIG. 3C). Of the most abundantly mutated lung cancers examined here, only 40% harbor the signature for tobacco smoke. We find that 35% of hypermutated lung have a high proportion of the UV light signature (>40% contribution) and 5% have evidence for alkylating agent-associated mutations (n=100). Furthermore, the UV light-associated signature was almost exclusively observed in the squamous cell carcinoma subtype (FIG. 11 ). This observation was previously reported for three hypermutant lung squamous cell carcinomas (Campbell et al., 2016). Either these are all misdiagnosed skin cancers with metastasis to the lung, as has previously been suggested, or perhaps, when hypermutated they form a distinct group based on a nucleotide context that reflects their cell of origin.

Sarcomas—tumours not previously associated with hypermutation—also clustered in an unexpected way. Hypermutated sarcomas were primarily in cluster C6—a large proportion of cases (70%) had high levels of UV-associated mutations (FIG. 3C). We validated this finding using available TOGA sarcoma data (exome sequence; FIG. 12 ), confirming that most hypermutated sarcomas bear the imprint of sun exposure. Indeed, in cluster C6 we see an enrichment for soft tissue angiosarcomas, a rare and aggressive vascular tumour that often presents cutaneously, usually on the face and scalp (Dossett et al., 2015). Soft tissue malignant peripheral nerve sheath tumour (MPNST), which can also arise cutaneously, were similarly enriched in C6. We then examined the pathology records from TOGA and, likewise, found that sarcomas with high UV-associated mutations were mostly superficial cancers. Thus, through this unbiased analysis, we have shown that UV light is associated with mutagenesis of mesenchymal cells, not just skin epithelia.

Alkylating agents, such as temozolomide, are known mutagens that leave a specific imprint on the genome. One of our clusters, C5, is dominated by brain cancers with overwhelming contribution of alkylating-associated mutations (on average 72%). We observed similar signatures in skin cancers (14% of the top most mutated skin cancers in this cohort), lung cancers, pancreatic cancers and leiomyosarcomas (FIG. 3C). Hypermutation should be therefore be considered in any relapsed cancers treated with alkylators, regardless of its cell of origin. This data reinforces the notion that a small set of processes drives hypermutation in a wide variety of tumour types.

Mutational Signatures Mark the History of Cancer Development

These data suggested that the signatures of hypermutation vary depending on the order of mutagen exposure. For example, the trinucleotide pattern of 01 resembles those of inherited gliomas in the CMMRD syndrome (Shlien et al., 2015) and would therefore suggest that these patients do in fact also harbor germline MMR mutations. If true, the overall trinucleotide composition of 01 represents an early constitutional MMR defect, followed by acquired secondary POLE, whereas tumours in cluster C3 have acquired POLE followed by MMR. To test this hypothesis directly, we sequenced a cohort of 1521 samples for which the temporal order of mutation was known (FIG. 4 ) from the international bMMRD consortium. This included: (1) cancers with germline MMR mutations plus either POLE, POLD1 mutations, or neither, as secondary event; (2) cancers arising in carriers of germline POLE mutations; and (3) gliomas whose hypermutation was due to temozolomide treatment.

We matched the nucleotide context of variants found in these childhood cancers, whose mutation order are known, to the clusters (FIG. 3 ), which were derived from an unannotated cohort of mostly adult cancers. Indeed, germline MMRD with secondary polymerase mutations matched cluster C1 whereas the cancers from patients with germline MMRD, such as Lynch or CMMRD without secondary polymerase deficiency matched cluster C2 (FIGS. 4A and B). C3 matched tumours from patients with germline POLE mutations. The difference between C2 and C3 suggests that MMR deficiency emerges early in the former and late in the latter. Therefore, in microsatellite stable tumours, ultra-hypermutation is driven by an early polymerase proofreading defect, while mismatch repair appears later. The timing of these events throughout the history of these cancers provides an explanation for the unexpected microsatellite stable phenotype observed in the POLE ultra-hypermutant, mismatch-repair deficient tumours seen here (FIG. 1D, FIG. 3B) and in other reports (Cancer Genome Atlas, 2012; Kandoth et al., 2013).

We next wanted to determine how the context of mutations changes over the course of each tumour's evolution. We summarized our observed mutation spectra using the established signature labels, as previously defined (Alexandrov et al., 2013). In this way, we ascribed specific causes to individual established signatures. We confirmed that “Signature 10” is associated with POLE exonuclease mutation. As expected, Signature 10 was high in those tumours where POLE was mutated early, from cluster C3 (e.g. germline POLE), and low in the late POLE tumours, seen in cluster C1. The mutation signature of POLD1 had not been previously described. We saw an enrichment of Signature 20 in cases with MMR and secondary POLD1. This is especially true for POLD1 L606M, a hotspot mutation in motif A of the polymerase domain. Significantly, Signatures 14 and 15 were enriched in all germline CMMRD hypermutant cancers, with much higher contribution than all other signatures. Gliomas with temozolomide-induced hypermutation were well matched to Signature 11. It was clear that many replication repair tumours present, at diagnosis, with a mix of signatures due to differing mutational histories. To separate out early and late signatures, we performed subclonal analysis (Methods). In childhood cases with MMR and secondary POLE (i.e. cluster C1), we do in fact see late-arising Signature 10, found exclusively in the subclone (FIG. 4C). Furthermore, high depth sequencing enabled us to detect subclones in multiple other cancers. For example, we see late-arising polymerase deficiency in a lung cancer, on the background of an abundance of smoking-associated mutations, and the late emergence of the treatment-associated alkylating agent signature in a skin cancer that is otherwise dominated by the UV light signature (FIGS. 4D, E and F).

Mutational Signatures Predict Germline Replication Repair Mutations

Finally, we tested whether somatic mutation burden combined with signatures—as determined by clinical panel sequencing—could reveal a germline cancer predisposition syndrome. We performed signature analysis on all ultra-hypermutant pediatric cancers and subsequently gathered clinical genetic information on most. As expected, all ultra-hypermutant cancers harbored mutations in either the MMR and/or DNA polymerases. Strikingly, a strong replication repair signature was observed in all tumours from patients with confirmed genetic diagnosis of germline MMR or POLE mutations (FIG. 5A). The only pediatric glioma that did not harbor the replication repair signature was found to have the alkylator signature (thus suggesting that the hypermutation was treatment induced, as seen in adults). Three tumours exhibited a small but significant alkylator signature. These were subsequently determined to be recurrent gliomas from CMMRD children whose treatment included temozolomide. In all cases, the initial biopsy did not reveal the alkylator signature, which further confirms the ability of mutational signatures to determine the natural history of cancer. These observations were also observed in hypermutant tumours with lower mutational burden (FIG. 5B).

Discussion

The mutations that accumulate during carcinogenesis leave imprints on the genome, and this is especially true in hypermutant cancers. Following these footprints can provide insight into tumour classification, help to pinpoint the true drivers that arose during the cancer's evolution—of which, some can even be traced back to the germline.

We found hypermutation in approximately 1 in 20 childhood cancers and 1 in 6 adult cancers. One sees an enrichment amongst tumours that have been subjected to continuous long term exposure to genotoxic agents, such as UV light in melanomas. For recurrent tumours, the treatment itself may be the primary cause of hypermutation. Exposure to chemotherapies such as alkylators or thiopurines can lead to replication repair deficiency (not unlike what occurs with germline predisposition or early somatic MMR mutations during initial tumour development) (Nguyen et al., 2014; Swann et al., 1996). These recurrent hypermutant cancers will be resistant to chemotherapy and other agents as they are driven by very different factors than the initial tumours. Knowledge of their mutational load and signatures could be used for targeted or immune based treatment strategies (Topalian et al., 2016) which are gaining success as cancer therapeutics, particularly in patients with high mutational load.

Driver mutations in POLE and POLD1 have only recently been described (Shinbrot et al., 2014). Our method for separating the drivers from the many passengers—the in vivo human mutagenesis screen—is likely the most direct way of measuring functional consequence of putative mutators (more so than conservation analysis or impact predictors such as PolyPhen and SIFT). Further research is required to fully understand why mutations in the polymerase domain are associated with hypermutation and putative loss of proofreading ability. A similar approach could be applied to other mutator phenotypes, driven by different genes, especially if associated with a unique signature (Scarpa et al., 2017).

Hypermutated tumours acquire their many mutations over an extended time period, many of which likely have no, or very little, functional consequence. In contrast, non hypermutated tumours are driven by a select few drivers, each contributing some significant pro-neoplastic property. Despite the high amount of noise, the “information content” and ability to infer tumour evolution from the multitude of passengers in a hypermutant tumour may, in many cases, be superior to that of a non hypermutated cancer. As seen here, these passengers (i.e. the “noise”), when considered together, bear the imprint of specific signatures that can differentiate clinically relevant subgroups (FIG. 3 ). For example, microsatellite stable but ultra-hypermutant cancers could be differentiated on the basis of mutational signature alone. As could squamous cell carcinomas of the lung that likely originated from metastatic skin cancers, and peripheral sarcomas with evidence for UV-induced damage.

Furthermore, in hypermutant cancers mutational signatures helped demarcate early from late emerging landmarks. This was seen in temozolomide-induced hypermutant gliomas amongst other cancers. Late-arising signatures of hypermutation can increase at recurrence, altering treatment options, even in the background of an already hypermutated cancer (e.g. driven by UV light or smoking, FIG. 4 ). Finally, in replication repair deficiency, one can use these signatures to trace the drivers to the germline. The cancer genome becomes a powerful new diagnostic aid for an underlying germline susceptibility. As one specific example, if a young patient is found to have a hypermutant tumour with a signature similar to cluster C1, their family should be offered genetic counseling and testing for CMMRD—a syndrome that is underdiagnosed due to the lack of clear clinical warning signs (Amayiri et al., 2016; Durno et al., 2015).

In summary, through the analysis of tumour samples from 81,337 children and adults we defined thresholds for hypermutation, revealing many tumour types whose signatures helped to reveal subgroups—a new taxonomy of hypermutation cancers defined by the type and order of mutagen exposure. This can be exploited for better patient management, including the identification of patients who may be most likely to benefit from the use of checkpoint inhibitors.

Development of a Tumour Classification System Based on New Signatures

Although Alexandrov and Stratton have performed foundational work on mutational signatures, using non-negative matrix factorization, their exploration of hypermutant cancers has been limited. These were mostly considered as exceptional outliers rather than studied in their own right. Our method uses the cancer genome's overall trinucleotide makeup to look for similarity within and between every istological type of hypermutant cancer. Using this unbiased approach, in which cancers aren't “fitted” to previously defined common signature types, we discovered several new signatures that delineate important clinical and biological cancer subgroups, regardless of tissue of origin. For example, we found three novel subtypes of replication repair deficient cancer. These only imperfectly correspond to the common signature vocabulary (i.e. Sanger signatures 6, 10, 14, 15 and/or 20). Instead, we find that the timing in which the patient was exposed to specific mutagens creates a unique combination of mutations in their tumour, and these allow for accurate biological and clinical stratification.

Uncovering the Clinical and Biological Impact of Mutation Bursts in Hypermutant Cancer

We have additionally show that the subgroups, defined by internal and external mutagenic events, display dramatic differences in temporal dynamics (i.e. tumour evolution). As seen in FIG. 13 , some tumour clusters accumulated mutations gradually, reaching hypermutation over an extended period, while others do so in dramatic bursts. Cancers with gradual mutation accumulation appeared to reach a mutation burden plateau, while explosive cancers (with bursts) did not.

Strikingly, these results are independent of the cancer's type and are instead determined by its mutagen exposure. UV light, smoking and MMR Cluster 2 resulted in only gradual mutation accumulation. Early-to-mid mutational bursts (caused by POLE mutations, Cluster 3) had a more benign outcome, however patients with tumours harboring late arising bursts of mutation—associated with complete replication repair deficiency or TMZ treatment (Clusters C1 and C5)—had significantly reduced survival.

Therefore, while the cancers studied here have all achieved the same minimal mutation threshold, we can now study the differing routes they took to reach hypermutation, and these have clear survival implications.

The Mutation Signature is a Robust Readout of the Life History of the Tumour

We retraced the evolutionary history of tumour development using the mutational signatures and burden found in the genome alone, and found that hypermutant tumours harbor clear landmarks, indicating when and how their mutations were acquired. To our knowledge, these have been missed in all previous reports.

These Results have Already Impacted the Management of Patients with Hypermutant Cancers

We show that the evolutionary history of a cancer can be traced to the germline. This is a striking clinical implication, highlighted in FIGS. 14 and 15 , showing a procedure for diagnosing cancer predisposition syndrome for a cohort of patients whose tumour sequence suggested an underlying germline mutation. This led us to contact the referring physicians and confirm the germline mutation. For these patients and their families, our results ultimately led to genetic testing and the following striking outcomes:

-   -   Immediate initiation of tumour surveillance and early tumour         detection;     -   Suggestion of therapies to avoid. Children with hypermutant         cancers should not be treated with common chemotherapies, such         as alkylators and thioguanines, as their tumours are inherently         resistant to those treatments (FIG. 5 ). This explains the poor         survival of hypermutated childhood glioblastomas treated with         temozolomide and leukemias treated with thioguanines. Thus, a         better understanding of the evolutionary history of tumour         development, as uniquely provided by our signature analysis, can         impact patient management by ruling out specific therapies. As         we have shown these signatures may arise late in tumour         development, reflecting a recently acquired resistance         mechanism;     -   Initiation of immune checkpoint inhibition therapy (FIG. 15 ).         We now also describe the striking treatment response data for         these same patients.

Taken together, these biological insights, which (1) reveal the causes of hypermutation in childhood cancer, (2) precisely define the key residues in the replicative polymerases that trigger hypermutation, (3) uncover a new classification scheme for replication repair deficient cancers, and (4) time the order of driver mutations, have extremely important clinical consequences. These data have already impacted the lives of multiple patients, who are now aware of their familial risk and are being seen by a genetic counselor, undergoing routine tumour surveillance, and are being offered rational therapy. Far from being a hypothetical clinical benefit, management for patients and families described in this manuscript has improved.

CONCLUSIONS

The landscape of hypermutation was analyzed using the DNA sequence of >81,000 adult and childhood cancers. This included individuals with hypermutation induced by chemotherapy, carcinogens, or established germline mutations. We uncovered common drivers of hypermutation across a wide range of tumour types, and identified clear driver mutations in recently described cancer genes—ultimately showing that these initiating events could be traced to the germline.

From these analyses of hypermutation across human cancer, several exciting observations emerged. These include:

The frequency of hypermutation is greater than previously appreciated, and extends to many new cancer types. We defined the first rational threshold for hyper and ultra-hypermutation in childhood and adult cancer. We found that nearly 1 in 20 childhood and 1 in 6 adults cancers are hypermutated across unexpected histotypes. This novel classification has immediate impact on patient stratification for clinical trials with immune checkpoint inhibition.

A precise definition of drivers in the DNA replicative polymerases, unveils key residues that trigger genome-wide hypermutation. Using hypermutation and signatures as a readout of replication-repair dysfunction, we built a model to validate driver mutations in the DNA polymerases. Specifically, we validated every known driver mutation in Polymerase and δ, from >3000 total reported variants, and found 11 new mutator residues.

1. The landscape of hypermutation across cancer reveals novel subgroups, showing that previous mutagen exposure can be inferred from the genome alone. Using unsupervised analyses, we grouped cancers into one of nine major categories. We discovered unexpected subgroupings—both within and between cancer types—such as ultra-hypermutated colorectal cancers that, strikingly, are microsatellite stable. We show that major mutagens, such as UV light and alkylators, act more broadly than previously thought.

2. The mutation signature is a robust readout of the life history of the tumour. Using nucleotide composition and subclonal analysis, we find that the order in which critical genes are mutated determines the mutational signature of the eventual tumour.

3. Identification of germline mutations can be determined from the cancer genome alone. Using this approach we identified patients with probable cancer susceptibility, then confirmed the presence of germline mutations after having contacted their physicians or families. This has already had clinically actionable relevance in terms of genetic counseling and cancer risk assessment to both patients and families.

Overall, our findings shed light on the extent and timing of common mechanisms for hyper-mutagenesis in human cancer. This data will immediately impact tumour classification for clinical trials and inform genetic testing and counselling.

In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required. In other instances, well-known electrical structures and circuits are shown in block diagram form in order not to obscure the understanding. For example, specific details are not provided as to whether the embodiments described herein are implemented as a software routine, hardware circuit, firmware, or a combination thereof.

Embodiments of the disclosure can be represented as a computer program product stored in a machine-readable medium (also referred to as a computer-readable medium, a processor-readable medium, or a computer usable medium having a computer-readable program code embodied therein). The machine-readable medium can be any suitable tangible, non-transitory medium, including magnetic, optical, or electrical storage medium including a diskette, compact disk read only memory (CD-ROM), memory device (volatile or non-volatile), or similar storage mechanism. The machine-readable medium can contain various sets of instructions, code sequences, configuration information, or other data, which, when executed, cause a processor to perform steps in a method according to an embodiment of the disclosure. Those of ordinary skill in the art will appreciate that other instructions and operations necessary to implement the described implementations can also be stored on the machine-readable medium. The instructions stored on the machine-readable medium can be executed by a processor or other suitable processing device, and can interface with circuitry to perform the described tasks.

The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

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All references referred to herein are expressly incorporated by reference in their entireties.

TABLE 4 Tumour samples analyzed using multiple platforms to demonstrate concordance in mutation burden. ID Panel WES WGS Mutation burden S1 X X X Low S2 X X X Medium S3 X X X High S4 X X X High S5 X X X High S6 X X X High S7 X X X High S8 X X X High S9 X X X High S10 X X X High S11 X X X High S12 X X X High S13 X X X High S14 X X X High S15 X X Medium S16 X X Medium S17 X X Medium S18 X X Medium S19 X X Medium S20 X X Medium S21 X X Low S22 X X Low S23 X X Low S24 X X Low S25 X X Low S26 X X Low S27 X X Low S28 X X Low S29 X X Low S30 X X Low S31 X X Low S32 X X Low S33 X X Low S34 X X Low S35 X X Low

TABLE 5 All unique variants identified in POLE and POLD1 tested for driver ability Associated mutation burdens are representative for each variant POLE M1L 2.7 POLE M1V 0.9 POLE M1V 17.1 POLE M1T 0.9 POLE M1V 3.6 POLE S2F 4.5 POLE S2F 15.3 POLE S2F 7.2 POLE R4K 52.3 POLE R4G 0.9 POLE G6S 1.8 POLE G6C 20.7 POLE G6S 1.8 POLE G6D 2.7 POLE G6S 0.9 POLE G7R 1.8 POLE G7L 6.3 POLE A11S 1.8 POLE D12N 12.6 POLE D12N 4.5 POLE D12E 0.9 POLE D12N 5.4 POLE D12N 27 POLE D12N 18.9 POLE A15T 17.1 POLE D16N 14.4 POLE D16N 27.9 POLE D16N 7.2 POLE G17A 2.7 POLE G17S 53.2 POLE G17D 68.5 POLE E18K 29.7 POLE E18Q 8.1 POLE E18* 9 POLE E18K 15.3 POLE E18K 16.2 POLE E18K 5.4 POLE E18* 49.5 POLE E18* 24.3 POLE E18Q 36 POLE E18* 29.7 POLE E18K 12.6 POLE E18K 42.3 POLE E18K 21.6 POLE E18* 44.1 POLE E18Q 19.8 POLE E18K 22.5 POLE E18Q 12.6 POLE E18K 5.4 POLE E18K 5.4 POLE E18K 27.9 POLE E18K 17.1 POLE E18K 14.4 POLE E18K 6.3 POLE E18K 6.3 POLE E18K 5.4 POLE E18K 11.7 POLE A19D 5.4 POLE S20I 109 POLE R21G 29.7 POLE D23G 4.5 POLE D23N 2.7 POLE D23G 6.3 POLE D23G 4.5 POLE D23N 33.3 POLE A25S 1.8 POLE A25V 132.4 POLE A25T 0 POLE A25S 0.9 POLE S27F 18 POLE S27F 11.7 POLE S28* 25.2 POLE S28L 9 POLE S28* 20.7 POLE S28L 6.3 POLE V29D 0.9 POLE S30L 45.9 POLE S30* 5.4 POLE S30L 7.2 POLE S30L 521.6 POLE S30L 703.6 POLE S30L 0 POLE L32R 2.7 POLE L32R 5.4 POLE R34C 7.2 POLE R34L 0.9 POLE R34H 13.5 POLE R34C 64.9 POLE R34L 1.8 POLE L35Q 18.9 POLE L35Q 1.8 POLE R37P 5.4 POLE R37Q 106.3 POLE R37W 3.6 POLE R37Q 12.6 POLE R37Q 17.1 POLE R37W 44.1 POLE R37Q 44.1 POLE S38N 5.4 POLE S38N 2.7 POLE S38N 8.1 POLE S38C 2.7 POLE S38C 3.6 POLE S38R 0.9 POLE Q39K 753.2 POLE W40C 12.6 POLE D42V 3.6 POLE D42V 0 POLE D42N 320.7 POLE D42V 1.8 POLE D42V 2.7 POLE K43N 16.2 POLE M44I 43.2 POLE M44V 5.4 POLE D45N 99.1 POLE R47L 9 POLE R47Q 4.5 POLE R47L 17.1 POLE R47Q 12.6 POLE R47Q 4.5 POLE G49D 9.9 POLE E51Q 21.6 POLE R52L 33.3 POLE R52L 38.7 POLE P56L 47.7 POLE P56S 753.2 POLE G57V 13.5 POLE K59N 9 POLE K59N 11.7 POLE G61D 5.4 POLE W62C 5.4 POLE W62* 8.1 POLE W62C 53.2 POLE I64V 58.6 POLE M66I 10.8 POLE P68A 16.2 POLE P68T 19.8 POLE P68L 209.9 POLE P68S 10.8 POLE T69S 6.3 POLE E70K 12.6 POLE E70K 6.3 POLE I71M 227.9 POLE I71V 49.5 POLE D73G 2.7 POLE E74K 18 POLE E74K 36.9 POLE D75E 3.6 POLE K76M 34.2 POLE R77C 69.4 POLE R77C 9 POLE R77L 9.9 POLE R77C 1.8 POLE G79V 2.7 POLE S80N 5.4 POLE A81T 1.8 POLE A81S 60.4 POLE A81T 34.2 POLE V82L 3.6 POLE D83N 85.6 POLE D83Y 39.6 POLE Y84C 0.9 POLE Y84H 22.5 POLE I87V 3.6 POLE I87V 9 POLE D89A 7.2 POLE D89A 3.6 POLE D90Y 36 POLE D90N 5.4 POLE G91R 0.9 POLE G91R 2.7 POLE G91E 3.6 POLE G91E 5.4 POLE G91E 0 POLE G91E 4.5 POLE G91R 2.7 POLE G91E 6.3 POLE G91E 3.6 POLE G91E 2.7 POLE S92G 2.7 POLE F94Y 89.2 POLE F94C 10.8 POLE K95N 78.4 POLE K95N 9.9 POLE A97T 4.5 POLE A97G 3.6 POLE K101I 1.8 POLE K101R 3.6 POLE K101E 7.2 POLE P102L 38.7 POLE P102S 147.7 POLE F104L 608.02 POLE F104L 37.8 POLE F104L 8.1 POLE F104I 9.9 POLE Y105F 6.3 POLE Y105F 7.2 POLE Y105F 14.4 POLE A107V 541.36 POLE A107V 608.02 POLE A107V 409.18 POLE A107V 41.4 POLE A107V 3.6 POLE A107V 26.1 POLE A107V 238.7 POLE A107S 43.2 POLE A107V 27.9 POLE R109T 24.3 POLE R109K 18 POLE R109T 41.4 POLE E113* 19.8 POLE E113D 29.7 POLE E113* 6.3 POLE R114Q 1.8 POLE R114* 0 POLE R114Q 0.9 POLE R114* 3.6 POLE E115K 155 POLE E115K 409 POLE E115K 0 POLE V116I 6.3 POLE V116A 65.8 POLE S118Y 117.1 POLE F119S 4.5 POLE F119L 2.7 POLE F119L 4.5 POLE F119L 6.3 POLE L120V 19.8 POLE L120V 0 POLE K122E 21.6 POLE K122N 23.4 POLE K122N 224.3 POLE K122N 19.8 POLE K123N 45.9 POLE Q125P 8.1 POLE Q125* 47.7 POLE Q125* 26.1 POLE Q125H 22.5 POLE Q125* 73 POLE K127I 82 POLE A129T 3.6 POLE K130E 13.5 POLE V131L 8.1 POLE V131L 0 POLE V131L 10.8 POLE V131L 14.4 POLE V131L 0.9 POLE E132Q 32.4 POLE V134I 4.5 POLE P135S 296 POLE P135S 3.6 POLE P135S 0 POLE P135S 8.1 POLE K136R 4.5 POLE E137K 4.5 POLE E137Q 38.7 POLE D138G 3.6 POLE D138E 4.5 POLE D138E 72.1 POLE L139V 35.1 POLE P142Q 112.6 POLE P142L 57.7 POLE N143S 3.6 POLE N143D 20.7 POLE N143H 0 POLE H144L 7.2 POLE L145F 9 POLE G147V 13.5 POLE G147C 7.2 POLE G147V 0 POLE L148F 0.9 POLE L148F 20.7 POLE K149R 35.1 POLE K149R 3.6 POLE R150* 20.7 POLE R150Q 245.9 POLE R150* 0.9 POLE R150* 117.1 POLE I153M 10.8 POLE I153M 2.7 POLE T159A 14.4 POLE E161* 37.8 POLE V164F 1.8 POLE K165R 16.2 POLE R167K 11.7 POLE R167W 26.1 POLE R167T 8.1 POLE E169D 21.6 POLE E169G 69.4 POLE I170M 11.7 POLE S171F 16.2 POLE P172L 62.2 POLE P172S 39.6 POLE V174M 11.7 POLE V174L 29.7 POLE V174L 1.8 POLE V174L 31.5 POLE N177S 1.8 POLE N177S 3.6 POLE R178M 3.6 POLE E179G 2.7 POLE H182R 36 POLE H182Y 130.6 POLE S184I 29.7 POLE D185N 4.5 POLE D185N 22.5 POLE D185N 5.4 POLE D185N 13.5 POLE D185N 3.6 POLE D185N 6.3 POLE D185G 41.4 POLE D185N 30.6 POLE A186T 5.4 POLE A186V 1.8 POLE A186T 2.7 POLE Y187* 4.5 POLE Y187C 10.8 POLE S192F 38.7 POLE S192F 7.2 POLE S193G 2.7 POLE S193G 4.5 POLE S193G 3.6 POLE Q196H 46.8 POLE Q196H 3.6 POLE Q196H 0 POLE R197S 26.1 POLE G198S 3.6 POLE G198S 7.2 POLE G198C 0.9 POLE G199C 13.5 POLE G199C 10.8 POLE V200I 2.7 POLE I201T 2.7 POLE I201T 9.9 POLE T202I 2.7 POLE E204K 18.9 POLE E205Q 36 POLE E206K 115.3 POLE T207I 386.5 POLE S208C 9 POLE K209N 3.6 POLE I211T 2.7 POLE I211T 4.5 POLE I211M 8.1 POLE A212P 12.6 POLE D216N 6.3 POLE D216G 2.7 POLE N217H 8.1 POLE V219M 1.8 POLE M221T 18 POLE M221T 2.7 POLE M221T 4.5 POLE R222H 19.8 POLE R222S 0 POLE R222C 71.2 POLE R222H 3.6 POLE E223K 62.2 POLE E223K 6.3 POLE Y224* 479.3 POLE Y224F 93.7 POLE D225H 18.9 POLE D225N 40.5 POLE V226I 27.9 POLE P227S 82 POLE P227S 140.5 POLE P227L 91 POLE P227L 2.7 POLE P227S 38.7 POLE P227S 634.2 POLE Y228C 17.1 POLE H229Y 5.4 POLE H229P 3.6 POLE R231L 5.4 POLE S233F 636.9 POLE I234V 1.8 POLE K237R 0.9 POLE K237R 13.5 POLE K237R 9 POLE K237R 9.9 POLE K237R 6.3 POLE I238N 180.2 POLE H239Y 119.8 POLE V240M 7.2 POLE V240M 67.6 POLE V240M 6.3 POLE R249* 130.6 POLE R249L 33.3 POLE R249Q 12.6 POLE R249P 18 POLE G250E 4.5 POLE G250E 9 POLE G250* 7.2 POLE G250E 52.3 POLE G250E 8.1 POLE N251Y 7.2 POLE N251Y 3.6 POLE F253L 7.2 POLE F253S 1.8 POLE P254L 3.6 POLE P254L 0.9 POLE P254L 14.4 POLE P254L 76.6 POLE V255I 17.1 POLE E256K 101.8 POLE I257T 5.4 POLE T258N 9.9 POLE R259C 9 POLE R259C 9.9 POLE R259C 0.9 POLE R259C 10.8 POLE R259C 0.9 POLE R260P 38.7 POLE R260* 15.3 POLE R260L 20.7 POLE D261N 9.9 POLE D262H 2.7 POLE D262H 0.9 POLE L263I 4.5 POLE R266* 3.6 POLE R266Q 104.5 POLE R266Q 3.6 POLE R266P 5.4 POLE D268N 63.1 POLE D268N 3.6 POLE V271I 5.4 POLE V271I 10.8 POLE V271I 227.9 POLE V271I 2.7 POLE F274S 1.8 POLE D275V 43.2 POLE D275V 17.1 POLE D275A 226.1 POLE D275G 318.9 POLE T278M 12.6 POLE T278M 68.5 POLE T278M 2.7 POLE T278M 37.8 POLE T278N 39.6 POLE K280N 4.5 POLE P286R 259.5 POLE P286R 493.7 POLE P286R 479.3 POLE P286R 394.6 POLE P286R 227.9 POLE P286R 162.2 POLE P286R 305.4 POLE P286R 123.4 POLE P286R 145.9 POLE P286S 42.3 POLE P286R 133.3 POLE P286R 342.3 POLE P286R 450.5 POLE P286R 216.2 POLE P286S 58.6 POLE P286R 245 POLE P286R 223.4 POLE P286R 155 POLE P286R 153.2 POLE P286R 84.7 POLE P286R 168.5 POLE P286R 163.1 POLE P286R 80.2 POLE P286R 100.9 POLE P286R 203.6 POLE P286R 226.1 POLE P286R 172.1 POLE P286R 208.1 POLE P286R 62.2 POLE P286H 212 POLE D287N 2.7 POLE D287N 30.6 POLE A288V 155.9 POLE A288V 578.4 POLE A288V 386.5 POLE E289* 24.3 POLE D291Y 71.2 POLE Q292K 1.8 POLE M294I 32.4 POLE M294I 17.1 POLE M294I 0 POLE M295I 180.2 POLE I296V 7.2 POLE S297F 218 POLE S297F 386.5 POLE S297F 39.6 POLE S297F 115.3 POLE Y298C 7.2 POLE Y298C 99.1 POLE M299V 0 POLE M299V 9 POLE I300N 9.9 POLE I300F 14.4 POLE D301G 3.6 POLE D301G 4.5 POLE G302D 48.6 POLE Q303H 311.7 POLE G304S 179.3 POLE Y305F 77.5 POLE Y305F 98.2 POLE L306F 226.1 POLE I307L 14.4 POLE N309S 2.7 POLE N309S 18 POLE R310S 8.1 POLE E311D 10.8 POLE I312T 12.6 POLE V313I 6.3 POLE S314* 5.4 POLE S314* 5.4 POLE S314* 18.9 POLE D316V 6.3 POLE D316N 4.5 POLE I317V 10.8 POLE E318K 9.9 POLE D319H 4.5 POLE F320C 1.8 POLE F320L 7.2 POLE F322L 20.7 POLE T323P 134.2 POLE T323A 168.5 POLE P324L 17.1 POLE P324F 92.8 POLE K325* 9 POLE E327V 433 POLE Y328C 0 POLE Y328C 0.9 POLE G330A 0 POLE C333F 9.9 POLE F335S 636.9 POLE E337K 5.4 POLE D339V 0 POLE D339V 15.3 POLE D339V 6.3 POLE D339V 3.6 POLE E340D 42.3 POLE E340K 139.6 POLE A341P 0.9 POLE A341T 236 POLE A341P 15.3 POLE H342Y 11.7 POLE L343V 7.2 POLE L343V 4.5 POLE Q345* 206.3 POLE Q345* 95.5 POLE Q345* 226.1 POLE W347C 2.7 POLE W347C 4.5 POLE W347S 45.9 POLE W347C 4.5 POLE E349Q 12.6 POLE E349K 7.2 POLE E349Q 13.5 POLE V351I 46.8 POLE V351F 4.5 POLE V351F 3.6 POLE V351I 6.3 POLE V351I 6.3 POLE V351I 0.9 POLE V351F 34.2 POLE V351I 0.9 POLE V351I 4.5 POLE V351I 2.7 POLE Q352P 5.4 POLE Q352P 4.5 POLE E353D 6.3 POLE T357I 17.1 POLE I358N 4.5 POLE M359I 14.4 POLE T361P 4.5 POLE T361A 3.6 POLE Y362C 5.4 POLE Y362C 7.2 POLE G364V 26.1 POLE G364R 9 POLE G364W 21.6 POLE D365N 5.4 POLE F366C 0.9 POLE F366L 208.1 POLE F367L 238.18 POLE F367L 472.56 POLE D368N 36 POLE F371L 3.6 POLE E373D 2.7 POLE A374G 7.2 POLE R375Q 1.8 POLE R375L 10.8 POLE R375W 16.2 POLE R375Q 0 POLE R375Q 2.7 POLE R375W 0.9 POLE H379Y 63.1 POLE G380S 3.6 POLE G380C 16.2 POLE G380C 19.8 POLE G380S 2.7 POLE G380S 3.6 POLE S382N 1.8 POLE S382T 4.5 POLE S382N 4.5 POLE M383V 1.8 POLE M383L 6.3 POLE Q385H 6.3 POLE Q385L 31.5 POLE E386K 9.9 POLE E386K 11.7 POLE E386K 1.8 POLE G388D 5.4 POLE Q390* 92.8 POLE Q390* 11.7 POLE D392G 59.5 POLE D392Y 9.9 POLE S393I 11.7 POLE S393N 11.7 POLE Q394K 26.1 POLE Q394R 2.7 POLE G395R 2.7 POLE G395V 10.8 POLE E396Q 0.9 POLE E396D 35.1 POLE E396K 55.9 POLE Y397* 7.2 POLE K398R 25.2 POLE A399V 107.2 POLE A399V 21.6 POLE A399G 8.1 POLE P400L 116.2 POLE Q401H 3.6 POLE Q401* 17.1 POLE C402F 123.4 POLE D406E 69.4 POLE C407Y 2.7 POLE L408F 108.1 POLE L408F 131.5 POLE R409S 37.8 POLE V411L 699 POLE V411L 77.5 POLE V411L 45 POLE V411L 39.6 POLE V411L 500 POLE V411L 104.5 POLE V411L 136 POLE V411L 371.2 POLE V411L 578.4 POLE V411L 345.9 POLE V411L 315.3 POLE V411L 100 POLE V411L 238.7 POLE V411L 120.7 POLE V411L 301.8 POLE V411L 636.9 POLE V411L 703.6 POLE V411L 650.5 POLE V411L 245.9 POLE V411L 116.2 POLE V411L 279.3 POLE V411L 309.9 POLE V411L 360.4 POLE V411L 187.4 POLE V411L 105.4 POLE V411L 240.5 POLE V411L 324.3 POLE K412N 9.9 POLE K412N 7.2 POLE R413K 95.5 POLE D414G 155 POLE S415I 19.8 POLE S415N 668.5 POLE S415G 1.8 POLE P418S 71.2 POLE P418L 9 POLE V419G 309.9 POLE G420S 12.6 POLE G420D 6.3 POLE G420D 122.5 POLE G420C 14.4 POLE H422Y 5.4 POLE H422R 10.8 POLE H422Y 4.5 POLE H422Y 17.1 POLE H422Y 1.8 POLE L424I 336 POLE L424I 336 POLE L424P 590 POLE L424I 47.7 POLE L424V 3.6 POLE L424I 160.4 POLE L424F 0.9 POLE L424I 154.1 POLE L424F 6.3 POLE L424V 91.9 POLE L424V 4.5 POLE L424V 19.8 POLE L424V 3.6 POLE L424H 9.9 POLE L424V 5.4 POLE L424V 6.3 POLE K425R 5.4 POLE K425R 135.1 POLE K425R 1.8 POLE K425R 6.3 POLE A426V 333.3 POLE A426V 5.4 POLE A427S 12.6 POLE A427V 56.8 POLE A427V 0 POLE A427V 4.5 POLE K431N 14.4 POLE P436S 302 POLE P436H 541.36 POLE P436S 318.06 POLE P436H 532 POLE P436H 532 POLE P436H 409.18 POLE P436H 359 POLE P436S 433 POLE P436S 195 POLE P436S 333.3 POLE P436T 5.4 POLE P436L 67.6 POLE P436R 493.7 POLE P436L 36 POLE P436L 69.4 POLE V437M 6.3 POLE V437M 2.7 POLE V437M 12.6 POLE E438K 46.8 POLE L439P 9 POLE D440N 2.7 POLE P441L 2.7 POLE P441L 29.7 POLE P441L 7.2 POLE M444K 91.9 POLE M444K 116.2 POLE M444K 32.4 POLE R446Q 4.5 POLE R446Q 0 POLE R446Q 7.2 POLE R446Q 1.8 POLE R446Q 1.8 POLE R446Q 13.5 POLE R446Q 5.4 POLE R446Q 3.6 POLE R446Q 8.1 POLE R446Q 2.7 POLE R446Q 0.9 POLE R446Q 9 POLE R446W 8.1 POLE R446Q 0.9 POLE R446Q 7.2 POLE R446Q 15.3 POLE R446W 1.8 POLE R446Q 4.5 POLE R446Q 0 POLE R446Q 4.5 POLE R446Q 1.8 POLE R446Q 5.4 POLE R446Q 1.8 POLE R446Q 1.8 POLE R446Q 3.6 POLE R446Q 2.7 POLE R446Q 1.8 POLE R446Q 4.5 POLE R446Q 0 POLE R446Q 27.9 POLE R446Q 92.8 POLE R446Q 5.4 POLE R446Q 1.8 POLE R446Q 4.5 POLE R446Q 0.9 POLE R446Q 32.4 POLE R446Q 2.7 POLE R446Q 2.7 POLE R446Q 13.5 POLE R446Q 2.7 POLE R446Q 21.6 POLE R446Q 6.3 POLE R446Q 3.6 POLE R446Q 3.6 POLE R446Q 3.6 POLE R446Q 23.4 POLE R446Q 0.9 POLE R446Q 4.5 POLE R446Q 4.5 POLE R446Q 21.6 POLE R446Q 4.5 POLE R446Q 8.1 POLE R446Q 1.8 POLE R446Q 5.4 POLE R446Q 2.7 POLE R446Q 1.8 POLE R446Q 9.9 POLE R446Q 2.7 POLE R446Q 6.3 POLE M447V 10.8 POLE M447I 9 POLE A448V 213.5 POLE T449M 9.9 POLE T449M 3.6 POLE T449M 3.6 POLE E450K 665.8 POLE P452T 9.9 POLE Q453R 22.5 POLE Q453E 11.7 POLE Q453E 2.7 POLE Q453E 5.4 POLE Q453E 20.7 POLE Q453E 7.2 POLE Q453* 0.9 POLE Q453R 8.1 POLE Q453E 3.6 POLE A456P 33.3 POLE A456P 226.1 POLE A456S 6.3 POLE A456P 200 POLE A456P 233.3 POLE A456P 69.4 POLE A456P 314.4 POLE A456P 373.9 POLE Y458H 85.02 POLE Y458H 342 POLE Y458C 13.5 POLE Y458C 753.2 POLE S459F 295 POLE S459F 102.7 POLE S459F 311.7 POLE S459F 36.9 POLE S459F 146.8 POLE S459F 117.1 POLE S459C 12.6 POLE V460M 15.3 POLE V460M 4.5 POLE S461P 208 POLE S461P 496.24 POLE S461L 69.4 POLE S461T 447.8 POLE D462Y 278.4 POLE D462H 6.3 POLE A463D 532 POLE A463D 359 POLE A463V 231.5 POLE A463V 55.9 POLE A463T 809 POLE A465T 238.18 POLE A465T 472.56 POLE A465V 214.4 POLE A465S 21.6 POLE A465S 9.9 POLE A465T 28.8 POLE A465T 0.9 POLE T466A 2.7 POLE T466I 155.9 POLE L469V 2.7 POLE M471V 3.6 POLE M471I 22.5 POLE M471V 4.5 POLE V474F 35.1 POLE V474I 7.2 POLE V474F 9.9 POLE P476S 59.5 POLE A480V 7.2 POLE A480V 55 POLE T483I 41.4 POLE T483I 51.4 POLE I485V 54.1 POLE I485M 1.8 POLE P486L 56.8 POLE P489L 155.9 POLE P489T 100 POLE P489L 226.1 POLE P489H 8.1 POLE D490E 5.4 POLE D490Y 26.1 POLE D490N 3.6 POLE D490G 14.4 POLE D490N 3.6 POLE D490N 2.7 POLE D490Y 36.9 POLE D490N 6.3 POLE E491K 2.7 POLE E491K 1.8 POLE E491K 5.4 POLE E491K 73 POLE E491K 1.8 POLE E491K 409 POLE E491K 2.7 POLE E491K 13.5 POLE V492M 138.7 POLE R494W 14.4 POLE K495N 7.2 POLE G496D 76.6 POLE G498D 98.2 POLE L500Q 7.2 POLE E502A 1.8 POLE L505P 342.3 POLE M506I 2.7 POLE M506V 1.8 POLE M506V 1.8 POLE M506V 1.8 POLE V507A 4.5 POLE V507A 1.8 POLE A509V 21.6 POLE A509T 8.1 POLE A509G 42.3 POLE H511Q 0.9 POLE N513D 4.5 POLE N513D 2.7 POLE I514V 2.7 POLE I514F 4.5 POLE P517L 2.7 POLE P517L 0.9 POLE P517L 14.4 POLE P517L 1.8 POLE P517L 147.7 POLE P517S 56.8 POLE N518D 13.5 POLE K519Q 4.5 POLE Q520R 8.1 POLE Q520R 6.3 POLE E521* 9 POLE Q522E 3.6 POLE E523K 7.2 POLE E523V 9 POLE F524L 1.8 POLE N525S 0.9 POLE N525H 2.7 POLE N525S 9 POLE N525H 3.6 POLE N525H 4.5 POLE N525H 2.7 POLE N525S 7.2 POLE K526R 0.9 POLE D529G 5.4 POLE G531R 36.9 POLE G531R 9 POLE G531R 0.9 POLE G531* 29.7 POLE V533M 6.3 POLE V533M 6.3 POLE V533M 15.3 POLE V533M 3.6 POLE V533M 35.1 POLE V533M 4.5 POLE V533M 20.7 POLE S536F 217.1 POLE E537K 13.5 POLE E537Q 22.5 POLE Y539C 14.4 POLE V540F 15.3 POLE V540F 9.9 POLE V540F 29.7 POLE H543Y 34.2 POLE H543P 23.4 POLE H543Y 53.2 POLE V544L 41.4 POLE V544M 9 POLE E545D 4.5 POLE E545Q 64.9 POLE L547F 48.6 POLE E548K 22.5 POLE E548K 1.8 POLE E548K 1.8 POLE S549A 9 POLE G550E 0 POLE G550E 70.3 POLE G550W 37.8 POLE V551I 35.1 POLE V551A 3.6 POLE R553C 22.5 POLE R553C 62.2 POLE R553C 27.9 POLE R553L 4.5 POLE R553C 3.6 POLE R553L 12.6 POLE S554G 4.5 POLE D555V 13.5 POLE P557S 9 POLE P557S 2.7 POLE P557S 81.1 POLE P557R 2.7 POLE P557T 42.3 POLE C558S 4.5 POLE C558Y 4.5 POLE R559Q 17.1 POLE R559W 3.6 POLE R559Q 142.3 POLE R559Q 1.8 POLE R559Q 43.2 POLE R559Q 14.4 POLE R559W 5.4 POLE M562I 18.9 POLE A566T 19.8 POLE F567L 4.5 POLE D568Y 46.8 POLE D568H 45 POLE F569L 8.1 POLE F569L 0.9 POLE F569C 6.3 POLE F569S 3.6 POLE Q572R 23.4 POLE R573L 23.4 POLE R573L 64 POLE R573Q 5.4 POLE R573L 14.4 POLE V574D 14.4 POLE E575Q 5.4 POLE E575Q 22.5 POLE K576E 18.9 POLE K576E 29.7 POLE K576Q 18 POLE H580R 31.5 POLE A581T 8.1 POLE A581T 2.7 POLE A581T 6.3 POLE A581V 9.9 POLE L582F 3.6 POLE E583Q 45 POLE E585K 5.4 POLE E585* 27 POLE E586K 9 POLE K587R 4.5 POLE P589S 28.8 POLE P589T 5.4 POLE P589S 13.5 POLE E591G 7.2 POLE E591K 9.9 POLE Q592R 27 POLE V593A 238.18 POLE V593A 472.56 POLE T594A 18 POLE T594I 4.5 POLE T594A 9.9 POLE N595S 3.6 POLE E597K 4.5 POLE E597G 1.8 POLE E598Q 69.4 POLE V599A 3.6 POLE V599M 116.2 POLE V599M 0.9 POLE C600Y 4.5 POLE C600S 6.3 POLE D601V 3.6 POLE D601V 4.5 POLE E602K 45.9 POLE E602A 5.4 POLE E602Q 5.4 POLE E602D 8.1 POLE E602K 11.7 POLE E602G 80.2 POLE I603T 3.6 POLE S609F 7.2 POLE S609P 51.4 POLE V613I 4.5 POLE V613I 46.8 POLE V613I 27 POLE V613I 11.7 POLE P614L 174.8 POLE R616H 3.6 POLE R616C 3.6 POLE R616H 24.3 POLE R616H 5.4 POLE I617N 196 POLE E618K 12.6 POLE E618K 47.7 POLE C619Y 4.5 POLE C619Y 27.9 POLE P620S 809 POLE L621P 1.8 POLE L621F 209.9 POLE I622V 10.8 POLE I622M 9 POLE I622V 6.3 POLE D626Y 9.9 POLE D626G 238.7 POLE V627M 15.3 POLE G628E 5.4 POLE A629T 0 POLE A629S 67.6 POLE A629V 18.9 POLE P632R 2.7 POLE N633H 3.6 POLE N633H 13.5 POLE I634V 409.18 POLE I634V 5.4 POLE I634V 2.7 POLE I634V 2.7 POLE T637I 4.5 POLE R639C 2.7 POLE R639H 45 POLE L640Q 3.6 POLE Q641H 21.6 POLE Q641H 12.6 POLE S643F 99.1 POLE M645I 9 POLE D647G 24.3 POLE D647V 1.8 POLE D647N 0.9 POLE E648K 0.9 POLE E648K 19.8 POLE E648K 8.1 POLE A649V 561.3 POLE A653S 5.4 POLE A653S 6.3 POLE A653S 46.8 POLE A653S 34.2 POLE A653S 4.5 POLE A653S 27.9 POLE N657S 3.6 POLE K658T 11.7 POLE P659R 9 POLE P659S 10.8 POLE R665W 12.6 POLE R665Q 0 POLE R665Q 5.4 POLE R665W 21.6 POLE R665W 1.8 POLE M667L 0 POLE A668T 2.7 POLE W669* 32.4 POLE W669L 122.5 POLE Q670* 101.8 POLE W671* 11.7 POLE R672S 4.5 POLE G673S 1.8 POLE E674A 109 POLE E674A 0.9 POLE E674K 5.4 POLE M676L 17.1 POLE M676I 27.9 POLE R680C 0 POLE R680H 7.2 POLE R680H 0.9 POLE R680H 7.2 POLE R680C 1.8 POLE R680C 1.8 POLE R680H 1.8 POLE R680L 7.2 POLE S681R 1.8 POLE E682K 7.2 POLE Y683C 159.5 POLE R685P 30.6 POLE R685L 18.9 POLE R685P 15.3 POLE Q689H 5.4 POLE Q689L 52.3 POLE Q689H 38.7 POLE L690V 3.6 POLE E693Q 20.7 POLE E693* 22.5 POLE E693* 9.9 POLE K694E 10.8 POLE F695V 6.3 POLE F695V 7.2 POLE F695S 50.5 POLE F695V 5.4 POLE F695L 3.6 POLE F695V 1.8 POLE P696R 11.7 POLE P697T 9.9 POLE P697H 10.8 POLE P697T 1.8 POLE P700A 16.2 POLE P700L 3.6 POLE P700Q 4.5 POLE P700S 48.6 POLE P700S 5.4 POLE P700S 96.4 POLE P700L 124.3 POLE P700L 5.4 POLE P700L 2.7 POLE P700S 4.5 POLE E701Q 34.2 POLE G702W 134.2 POLE G702W 18.9 POLE G702R 14.4 POLE P703L 23.4 POLE A704S 218 POLE A704P 3.6 POLE R705W 9.9 POLE R705Q 12.6 POLE R705G 28.8 POLE H708Y 665.8 POLE H708R 13.5 POLE E709K 4.5 POLE E709K 115.3 POLE R712C 45.9 POLE R712C 48.6 POLE R712C 44.1 POLE R712C 89.2 POLE R712G 3.6 POLE E713* 31.5 POLE E713K 0.9 POLE E714K 4.5 POLE E714K 2.7 POLE Q715R 8.1 POLE A716V 532 POLE E719K 62.2 POLE E719K 0 POLE E719K 67.6 POLE L723V 44.1 POLE A724T 12.6 POLE D725Y 1.8 POLE D725H 29.7 POLE Y726F 15.3 POLE Y726S 27.9 POLE R728W 0 POLE R728W 3.6 POLE R728W 45.9 POLE R728P 20.7 POLE R728W 2.7 POLE R728W 28.8 POLE R728Q 27.9 POLE K729I 100.9 POLE Y731F 16.2 POLE K732R 9.9 POLE K733N 69.4 POLE H735Y 78.4 POLE K738N 41.4 POLE K738N 1.8 POLE V739M 116.2 POLE E740K 20.7 POLE E740* 90.1 POLE E741G 10.8 POLE E741K 84.7 POLE E741G 3.6 POLE E741* 2.7 POLE E741G 1.8 POLE R742C 318.06 POLE R742C 96.4 POLE L743V 19.8 POLE T744A 55 POLE T744A 72.1 POLE T745A 20.7 POLE I746V 6.3 POLE I746T 35.1 POLE C747F 37.8 POLE Q748L 5.4 POLE R749Q 4.5 POLE R749W 56.8 POLE R749Q 2.7 POLE R749Q 3.6 POLE S752F 6.3 POLE F753L 92.8 POLE F753L 23.4 POLE Y754C 6.3 POLE Y754C 0.9 POLE V755M 114.4 POLE V755M 71.2 POLE V758M 532 POLE V758G 1.8 POLE V758M 114.4 POLE R759C 81.1 POLE R759H 0 POLE R759C 2.7 POLE R759C 5.4 POLE R759H 3.6 POLE A760V 98.2 POLE R762W 2.7 POLE R762W 3.6 POLE R762W 3.6 POLE R764M 46.8 POLE R765H 1.8 POLE R765C 13.5 POLE R765C 36.9 POLE R765C 314.4 POLE E767K 7.2 POLE F768V 109 POLE G770R 10.8 POLE V774G 6.3 POLE W775* 5.4 POLE W775* 93.7 POLE K776R 4.5 POLE K776R 5.4 POLE K776R 4.5 POLE V777E 195 POLE K777N 3.6 POLE K778T 1.8 POLE K778T 5.4 POLE L779F 99.1 POLE S780L 91.9 POLE S780L 9 POLE S780L 4.5 POLE S780L 1.8 POLE S780L 2.7 POLE S780L 447.8 POLE A781S 9 POLE A781V 52.3 POLE A781S 26.1 POLE A781T 145.9 POLE A782V 15.3 POLE V783M 2.7 POLE V783M 6.3 POLE V783M 11.7 POLE V783M 12.6 POLE V783L 5.4 POLE 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M829I 1.8 POLE E830K 6.3 POLE A832T 590 POLE I834T 45.9 POLE A840T 33.3 POLE A840G 69.4 POLE N841D 31.5 POLE N841D 2.7 POLE N841D 51.4 POLE I843V 4.5 POLE I843V 27 POLE T844I 368.5 POLE T844I 16.2 POLE Q845H 14.4 POLE A846E 148.6 POLE R847W 3.6 POLE E848D 25.2 POLE E848D 6.3 POLE I850T 5.4 POLE I850M 7.2 POLE I850M 3.6 POLE I850M 1.8 POLE E851* 8.1 POLE Q852R 120.7 POLE G854V 14.4 POLE R855W 8.1 POLE E858K 106.3 POLE E858* 251.4 POLE T861I 49.5 POLE D862G 1.8 POLE V867L 6.3 POLE L868V 10.8 POLE N870S 0 POLE S871C 48.6 POLE S871T 3.6 POLE S871N 121.6 POLE S871C 35.1 POLE P873Q 23.4 POLE P873L 5.4 POLE E874K 31.5 POLE K879N 2.7 POLE K879Q 9 POLE T880M 4.5 POLE T880M 1.8 POLE T880M 4.5 POLE T880L 0 POLE T880L 9 POLE T880K 19.8 POLE T880A 1.8 POLE T880M 66.7 POLE T880M 89.2 POLE T881I 224.3 POLE N882H 4.5 POLE K884R 1.8 POLE P886H 45.9 POLE P886L 32.4 POLE V888A 10.8 POLE S891F 236 POLE P893S 4.5 POLE P893L 1.8 POLE A895V 47.7 POLE A895V 3.6 POLE A895V 0 POLE A895V 0.9 POLE A895S 9 POLE M896V 9.9 POLE N898S 19.8 POLE I899V 18.9 POLE K902N 15.3 POLE K902N 88.3 POLE E903K 9 POLE E903D 6.3 POLE G904V 6.3 POLE G904A 8.1 POLE T906I 104.5 POLE Q909R 3.6 POLE Q911P 10.8 POLE E912Q 9 POLE E912Q 9.9 POLE E912* 17.1 POLE E912Q 17.1 POLE E912K 3.6 POLE E912K 13.5 POLE A914P 15.3 POLE E915K 82 POLE E915Q 27.9 POLE L919P 3.6 POLE L919F 0 POLE V922I 46.8 POLE R924G 4.5 POLE R924H 29.7 POLE E926Q 4.5 POLE S928N 0 POLE I929V 2.7 POLE F930C 4.5 POLE E932K 8.1 POLE V933I 138.7 POLE D934H 60.4 POLE D934H 47.7 POLE D934V 3.6 POLE G935M 27 POLE P936S 202.7 POLE L938F 0 POLE L938F 173.9 POLE L938V 12.6 POLE L942F 182.9 POLE L942F 500 POLE P943S 9 POLE P943S 150.5 POLE P943L 5.4 POLE S945F 320.7 POLE S945F 24.3 POLE E948* 160.4 POLE E948Q 69.4 POLE E948Q 9.9 POLE E948K 53.2 POLE R955M 8.1 POLE N960S 6.3 POLE N960S 6.3 POLE N960S 18.9 POLE N960S 3.6 POLE N960S 1.8 POLE N960S 6.3 POLE D962N 75.7 POLE D962G 9 POLE D962N 30.6 POLE G963C 30.6 POLE G963D 5.4 POLE G963S 29.7 POLE G963C 60.4 POLE G963D 2.7 POLE G963D 5.4 POLE S964F 21.6 POLE A966P 28.8 POLE A966S 19.8 POLE G970S 4.5 POLE G970S 7.2 POLE E972G 3.6 POLE E972Q 30.6 POLE R975L 7.2 POLE R975C 31.5 POLE R976H 8.1 POLE R976H 42.3 POLE R976H 31.5 POLE R976C 8.1 POLE R976C 2.7 POLE G977R 5.4 POLE E978G 608.02 POLE E978* 15.3 POLE E978G 26.1 POLE E978K 80.2 POLE K983N 7.2 POLE K983N 69.4 POLE K983N 251.4 POLE Q986* 25.2 POLE S987F 1.8 POLE V989L 22.5 POLE V989L 3.6 POLE F990C 301.8 POLE E991Q 20.7 POLE G996D 18 POLE G996S 64 POLE G996D 145.9 POLE G996S 178.4 POLE T998M 8.1 POLE T998M 46.8 POLE T998M 10.8 POLE T998M 3.6 POLE T998M 1.8 POLE E1001Q 28.8 POLE V1002M 3.6 POLE V1002A 91.9 POLE S1005F 7.2 POLE D1011Y 34.2 POLE W1013* 57.7 POLE D1015N 159.5 POLE Y1018H 5.4 POLE Y1018C 62.2 POLE S1019I 0.9 POLE S1019C 15.3 POLE S1019I 6.3 POLE K1020R 9.9 POLE A1021E 4.5 POLE A1021S 3.6 POLE A1021G 2.7 POLE M1024V 0.9 POLE M1024I 0.9 POLE M1024I 9 POLE D1026H 18.9 POLE D1026H 11.7 POLE D1026N 35.1 POLE S1027A 2.7 POLE S1027C 20.7 POLE F1030Y 4.5 POLE F1030L 1.8 POLE E1031D 26.1 POLE E1031K 3.6 POLE I1033F 7.2 POLE R1037H 608.02 POLE R1037C 6.3 POLE R1037H 3.6 POLE R1037C 6.3 POLE S1038A 136 POLE M1039T 15.3 POLE R1041Q 8.1 POLE R1041W 5.4 POLE R1041Q 65.8 POLE K1042R 10.8 POLE E1048K 1.8 POLE E1048K 665.8 POLE Q1049H 6.3 POLE S1051P 3.6 POLE T1052A 0.9 POLE T1052M 2.7 POLE T1052M 9 POLE I1054V 2.7 POLE A1057V 336 POLE A1057V 336 POLE A1057V 0.9 POLE R1059C 2.7 POLE R1059L 18 POLE R1059C 24.3 POLE R1059C 309.9 POLE R1059C 318.9 POLE R1059C 46.8 POLE A1061V 118 POLE E1062K 51.4 POLE F1063L 60.4 POLE G1065E 46.8 POLE D1066E 1.8 POLE D1066N 8.1 POLE D1066N 0.9 POLE D1066N 0 POLE D1071H 32.4 POLE A1072T 0.9 POLE A1072G 3.6 POLE S1075C 0.9 POLE S1075C 8.1 POLE R1077H 218 POLE R1077H 3.6 POLE R1077H 17.1 POLE R1077H 3.6 POLE R1077H 17.1 POLE R1077L 28.8 POLE R1077H 12.6 POLE R1077H 32.4 POLE Y1078F 12.6 POLE K1083Q 12.6 POLE K1083Q 1.8 POLE P1084L 182 POLE E1085K 0.9 POLE E1085D 9.9 POLE E1085K 3.6 POLE E1085K 14.4 POLE E1085* 18 POLE E1085K 3.6 POLE E1085K 1.8 POLE G1086C 33.3 POLE T1090M 60.4 POLE T1090R 3.6 POLE E1091K 8.1 POLE E1091K 16.2 POLE E1091K 34.2 POLE R1092S 1.8 POLE R1092K 78.4 POLE A1093V 109.9 POLE P1095S 164 POLE L1096V 1.8 POLE P1103H 84.7 POLE T1104M 6.3 POLE T1104M 1.8 POLE T1104M 2.7 POLE T1104M 25.2 POLE T1104M 55.9 POLE V1105L 27.9 POLE V1105M 76.6 POLE K1107N 57.7 POLE F1109Y 119.8 POLE F1109C 6.3 POLE L1110I 3.6 POLE L1110I 35.1 POLE R1111Q 5.4 POLE R1111Q 8.1 POLE R1111P 18.9 POLE R1111Q 15.3 POLE R1111W 81.1 POLE R1111W 15.3 POLE W1113* 668.5 POLE W1113* 22.5 POLE L1114F 7.2 POLE K1115N 60.4 POLE K1115E 11.7 POLE K1115N 3.6 POLE S1116R 699 POLE S1118A 0 POLE S1118A 3.6 POLE S1118F 51.4 POLE D1121G 3.6 POLE D1123H 10.8 POLE I1124F 8.1 POLE R1125* 146.8 POLE R1125* 3.6 POLE A1126V 2.7 POLE A1126V 3.6 POLE A1126V 6.3 POLE I1127T 10.8 POLE L1128M 5.4 POLE D1129G 4.5 POLE W1130C 9 POLE W1130* 92.8 POLE D1131N 0.9 POLE D1131E 3.6 POLE D1131G 41.4 POLE Y1133C 5.4 POLE Y1133H 8.1 POLE I1134F 21.6 POLE E1135D 55 POLE E1135K 28.8 POLE R1136W 3.6 POLE R1136W 4.5 POLE R1136W 69.4 POLE R1136Q 15.3 POLE G1138* 45 POLE I1141M 8.1 POLE K1143N 6.3 POLE K1143N 46.8 POLE T1146I 334.2 POLE I1147V 21.6 POLE P1148S 10.8 POLE A1149V 52.3 POLE A1149V 36 POLE A1149V 35.1 POLE A1149V 4.5 POLE A1150T 77.5 POLE A1150T 0.9 POLE Q1152* 76.6 POLE Q1153* 2.7 POLE Q1153* 4.5 POLE K1155N 18.9 POLE K1155N 5.4 POLE P1157S 433 POLE P1157S 4.5 POLE P1157S 2.7 POLE P1159S 6.3 POLE R1160H 7.2 POLE R1160H 0.9 POLE R1160H 1.8 POLE P1164L 86.5 POLE P1164L 5.4 POLE D1165N 3.6 POLE D1165N 85.6 POLE D1165N 118.9 POLE K1170* 5.4 POLE L1172V 9 POLE E1173G 11.7 POLE D1176V 7.2 POLE K1179R 28.8 POLE K1179R 4.5 POLE Q1180H 9.9 POLE S1184G 6.3 POLE E1190K 12.6 POLE E1190G 23.4 POLE E1190Q 10.8 POLE G1191C 9 POLE R1192S 5.4 POLE R1192G 37.8 POLE R1193I 12.6 POLE R1193K 9.9 POLE R1193K 5.4 POLE R1193* 33.3 POLE R1193G 16.2 POLE R1193K 7.2 POLE Q1194R 1.8 POLE V1195I 561.3 POLE V1195I 2.7 POLE V1195F 22.5 POLE V1195D 11.7 POLE T1196M 1.8 POLE T1196M 75.7 POLE T1196M 43.2 POLE T1196M 7.2 POLE A1198V 73 POLE A1198D 5.4 POLE E1199K 7.2 POLE E1199K 6.3 POLE E1199D 70.3 POLE E1199G 31.5 POLE E1199K 9.9 POLE E1199K 7.2 POLE E1199K 17.1 POLE A1200T 809 POLE D1203N 69.4 POLE D1203Y 6.3 POLE S1204R 64 POLE S1204R 2.7 POLE P1205L 3.6 POLE P1205S 665.8 POLE P1205L 12.6 POLE P1205L 12.6 POLE R1206M 5.4 POLE R1206M 17.1 POLE P1207L 8.1 POLE P1207L 4.5 POLE P1207L 11.7 POLE P1207R 2.7 POLE S1208R 4.5 POLE D1211N 72.1 POLE M1212R 2.7 POLE D1214Y 18 POLE G1216S 0 POLE G1216C 52.3 POLE G1216C 7.2 POLE G1216D 3.6 POLE G1216S 18.9 POLE V1218I 2.7 POLE V1218L 5.4 POLE K1219Q 23.4 POLE H1222Y 6.3 POLE H1222Y 91.9 POLE A1224T 278.4 POLE A1225V 9.9 POLE P1226L 590 POLE P1226A 9.9 POLE P1226L 12.6 POLE V1227F 5.4 POLE T1228A 10.8 POLE T1228S 12.6 POLE K1232T 2.7 POLE R1233* 214.4 POLE R1233* 2.7 POLE R1233* 0 POLE L1235F 88.3 POLE L1235I 5.4 POLE W1236L 88.3 POLE E1237D 6.3 POLE S1238I 208 POLE S1238T 14.4 POLE S1238T 0.9 POLE Q1239H 6.3 POLE E1240Q 6.3 POLE E1240Q 66.7 POLE E1240* 54.1 POLE E1241Q 7.2 POLE S1242F 4.5 POLE S1242C 8.1 POLE S1242C 2.7 POLE S1242T 118 POLE S1242T 4.5 POLE S1242C 27 POLE S1242F 2.7 POLE S1242F 3.6 POLE S1242F 5.4 POLE D1244Y 36 POLE T1246M 70.3 POLE T1246R 2.7 POLE T1246M 5.4 POLE P1247S 25.2 POLE T1248P 5.4 POLE V1249M 21.6 POLE E1253Q 1.8 POLE I1254V 2.7 POLE G1256W 20.7 POLE Q1257L 8.1 POLE Q1257P 20.7 POLE A1260S 145.9 POLE A1260S 29.7 POLE G1262* 65.8 POLE G1262L 21.6 POLE G1262E 19.8 POLE Q1265* 36.9 POLE E1266A 32.4 POLE W1268* 110.8 POLE V1270G 160.4 POLE V1270I 1.8 POLE V1270F 32.4 POLE V1270F 6.3 POLE R1273W 235.1 POLE R1273L 27.9 POLE R1273W 74.8 POLE R1273W 5.4 POLE F1274L 51.4 POLE K1276N 66.7 POLE W1279L 110.8 POLE Q1280L 14.4 POLE Q1282L 22.5 POLE R1284W 2.7 POLE R1284G 1.8 POLE R1284W 4.5 POLE R1286C 2.7 POLE R1286C 650.5 POLE R1286C 64 POLE A1288V 3.6 POLE A1288V 3.6 POLE A1288P 39.6 POLE R1289C 41.4 POLE R1289H 3.6 POLE R1289L 20.7 POLE R1289C 40.5 POLE R1289L 29.7 POLE R1289S 4.5 POLE R1289H 4.5 POLE R1289C 3.6 POLE K1291N 3.6 POLE Q1293* 73 POLE L1295M 3.6 POLE L1295M 3.6 POLE E1296Q 29.7 POLE S1297X 218 POLE S1297L 10.8 POLE S1297L 4.5 POLE S1297L 22.5 POLE S1297L 59.5 POLE S1297L 5.4 POLE S1297L 7.2 POLE E1299K 10.8 POLE E1299Q 12.6 POLE V1301A 8.1 POLE L1302I 208 POLE L1302P 14.4 POLE R1303M 38.7 POLE G1305E 1.8 POLE G1305E 4.5 POLE G1305E 4.5 POLE G1305W 10.8 POLE G1305V 28.8 POLE A1306D 152.3 POLE A1306T 561.3 POLE A1306T 1.8 POLE A1306T 64.9 POLE I1307V 1.8 POLE I1307F 28.8 POLE I1307V 16.2 POLE R1308Q 145.9 POLE R1308L 4.5 POLE D1309G 6.3 POLE D1309N 8.1 POLE D1309N 251.4 POLE D1309N 1.8 POLE T1313M 8.1 POLE G1314W 32.4 POLE L1315M 25.2 POLE G1316E 114.4 POLE G1316W 5.4 POLE G1316E 12.6 POLE S1317C 9.9 POLE F1318S 636.9 POLE F1318L 25.2 POLE L1319F 4.5 POLE L1319V 64 POLE L1319S 9.9 POLE R1320Q 8.1 POLE R1320Q 2.7 POLE R1320Q 1.8 POLE R1320Q 20.7 POLE R1320* 111.7 POLE R1320* 18 POLE R1320* 318.9 POLE R1321K 18 POLE T1322N 4.5 POLE R1324C 4.5 POLE R1324C 1.8 POLE I1326V 5.4 POLE I1326V 18.9 POLE P1330L 152.3 POLE P1330L 11.7 POLE P1330S 113.5 POLE P1330L 24.3 POLE P1330L 134.2 POLE P1330L 44.1 POLE P1330L 450.5 POLE W1331C 5.4 POLE W1331* 193.7 POLE W1331L 10.8 POLE Q1332R 1.8 POLE I1333T 96.4 POLE Q1335* 228.8 POLE Q1335H 4.5 POLE I1336M 8.1 POLE E1338K 1.8 POLE E1338K 18 POLE E1338K 47.7 POLE E1338K 12.6 POLE Q1341H 44.1 POLE A1342S 20.7 POLE A1342T 10.8 POLE G1343S 21.6 POLE L1344P 0.9 POLE F1345L 24.3 POLE R1346K 313.5 POLE W1348* 3.6 POLE W1348C 26.1 POLE A1349V 608.02 POLE A1349V 6.3 POLE A1349V 116.2 POLE A1349V 1.8 POLE V1351L 2.7 POLE V1351I 0.9 POLE V1351I 5.4 POLE V1351I 79.3 POLE V1351I 13.5 POLE V1351I 9.9 POLE S1353R 4.5 POLE D1354E 8.1 POLE H1356D 0.9 POLE H1356D 8.1 POLE H1356D 0 POLE I1358T 40.5 POLE R1359S 27 POLE L1360Q 2.7 POLE S1361G 196 POLE S1361N 27 POLE S1361I 333.3 POLE P1363L 9 POLE R1364C 3.6 POLE R1364H 1.8 POLE R1364H 1.8 POLE R1364C 7.2 POLE R1364H 2.7 POLE R1364C 56.8 POLE R1364C 1.8 POLE R1364C 60.4 POLE R1364C 8.1 POLE R1364H 1.8 POLE R1364L 4.5 POLE F1366L 134.2 POLE F1366L 20.7 POLE Y1367H 9 POLE Y1367H 5.4 POLE Y1367D 5.4 POLE V1368M 0.9 POLE N1369S 3.6 POLE N1369S 10.8 POLE N1369D 131.5 POLE Q1370R 0.9 POLE Q1370* 71.2 POLE R1371Q 0 POLE R1371* 5.4 POLE R1371* 371.2 POLE R1371L 0 POLE R1371L 43.2 POLE R1371* 1.8 POLE R1371Q 1.8 POLE R1371Q 20.7 POLE V1372I 4.5 POLE A1373V 91.9 POLE A1373V 4.5 POLE A1375S 0.9 POLE A1375V 3.6 POLE A1375T 75.7 POLE E1376G 118.9 POLE E1376D 12.6 POLE E1377K 320.7 POLE G1378D 3.6 POLE A1379T 3.6 POLE S1380L 96.4 POLE S1380L 55.9 POLE S1380L 235.1 POLE S1380L 3.6 POLE S1380L 634.2 POLE Y1381F 18 POLE R1382C 98.2 POLE R1382C 39.6 POLE R1382C 6.3 POLE R1382S 1.8 POLE R1382L 2.7 POLE R1382H 2.7 POLE R1382H 8.1 POLE R1382H 34.2 POLE R1382C 100 POLE R1382C 2.7 POLE R1382C 636.9 POLE R1382H 2.7 POLE K1383* 16.2 POLE K1383N 36.9 POLE K1383N 8.1 POLE R1386W 112.6 POLE R1386Q 0.9 POLE R1386Q 75.7 POLE R1386W 145.9 POLE R1386W 7.2 POLE R1386G 17.1 POLE R1386W 45.9 POLE R1386Q 5.4 POLE V1387A 8.1 POLE V1387A 0.9 POLE P1389S 18.9 POLE P1389S 91 POLE R1390C 58.6 POLE R1390L 15.3 POLE R1390L 8.1 POLE R1390C 3.6 POLE R1390C 224.3 POLE R1390C 18 POLE R1390C 4.5 POLE R1390C 9 POLE R1390C 13.5 POLE R1390L 9.9 POLE S1391C 31.5 POLE S1391C 5.4 POLE S1391F 98.2 POLE S1391C 6.3 POLE M1393V 40.5 POLE M1393I 25.2 POLE Y1395H 3.6 POLE L1397R 0 POLE Y1398C 7.2 POLE E1399K 12.6 POLE E1399Q 5.4 POLE Y1400C 0.9 POLE V1402M 0.9 POLE P1403S 231.5 POLE P1403S 22.5 POLE E1404D 14.4 POLE E1404K 20.7 POLE M1406V 5.4 POLE M1406V 1.8 POLE Q1408* 9.9 POLE H1410P 0 POLE I1411V 7.2 POLE I1411L 4.5 POLE N1412Y 95.5 POLE E1417Q 16.2 POLE E1417K 8.1 POLE A1420S 6.3 POLE P1421L 1.8 POLE P1421T 6.3 POLE E1424K 5.4 POLE E1424K 2.7 POLE E1424K 6.3 POLE E1424K 1.8 POLE E1424K 8.1 POLE G1425C 4.5 POLE G1425F 34.2 POLE V1426L 21.6 POLE E1428K 22.5 POLE E1428Q 3.6 POLE E1428Q 8.1 POLE T1429S 3.6 POLE T1429S 4.5 POLE T1429S 4.5 POLE T1429S 4.5 POLE T1429S 26.1 POLE Q1430P 0 POLE P1432L 8.1 POLE P1432L 209 POLE P1432S 130.6 POLE P1432L 4.5 POLE P1432S 60.4 POLE P1432T 447.8 POLE P1432L 4.5 POLE P1432S 56.8 POLE P1432S 64.9 POLE P1432S 143.2 POLE P1432L 318.9 POLE P1432L 14.4 POLE F1435L 386.5 POLE R1436W 0 POLE R1436L 9 POLE R1436W 0.9 POLE R1436W 5.4 POLE A1437T 1.8 POLE A1437T 7.2 POLE H1440R 64 POLE G1442S 1.8 POLE V1444E 17.1 POLE V1444A 7.2 POLE V1444M 164.9 POLE V1444L 12.6 POLE V1447I 0 POLE V1447F 82.9 POLE Q1450H 2.7 POLE R1453S 18.9 POLE R1453K 561.3 POLE H1454N 112.6 POLE G1457D 25.2 POLE W1458* 4.5 POLE W1458* 53.2 POLE E1461Q 29.7 POLE E1461* 26.1 POLE A1464V 479.3 POLE E1466Q 4.5 POLE E1466K 1.8 POLE E1466Q 15.3 POLE H1467L 12.6 POLE H1467N 2.7 POLE H1467Y 18.9 POLE L1468V 5.4 POLE E1469Q 251.4 POLE R1471H 1.8 POLE R1471C 6.3 POLE R1471C 114.4 POLE R1471H 94.6 POLE R1471C 43.2 POLE R1471C 9 POLE R1471C 38.7 POLE R1471H 21.6 POLE R1471C 72.1 POLE R1471L 27 POLE R1471C 0.9 POLE S1472Y 336 POLE S1472Y 336 POLE S1472F 2.7 POLE S1472T 3.6 POLE S1472F 25.2 POLE S1472F 33.3 POLE F1476C 2.7 POLE F1476C 4.5 POLE E1480K 7.2 POLE G1482R 7.2 POLE S1483G 145.9 POLE I1484V 0.9 POLE I1484V 2.7 POLE R1485C 2.7 POLE R1485H 6.3 POLE R1485H 3.6 POLE R1485C 12.6 POLE R1485C 38.7 POLE R1485H 20.7 POLE R1485C 20.7 POLE R1485C 9 POLE I1487S 9 POLE H1491N 39.6 POLE H1492R 36.9 POLE H1492Q 9 POLE H1492Y 2.7 POLE H1492L 38.7 POLE H1492Q 7.2 POLE Q1494E 25.2 POLE Q1494E 40.5 POLE Q1494E 4.5 POLE A1495V 0.9 POLE H1496Q 18.9 POLE K1497E 2.7 POLE K1497R 3.6 POLE K1497R 2.7 POLE K1497R 0.9 POLE G1501R 91 POLE G1501W 7.2 POLE I1502V 56.8 POLE I1502N 26.1 POLE F1503C 445.9 POLE F1503L 355.9 POLE I1504F 0.9 POLE I1504V 355.9 POLE P1505S 295 POLE P1505S 2.7 POLE P1505S 1.8 POLE P1505A 52.3 POLE P1505L 31.5 POLE P1505S 31.5 POLE P1505S 100.9 POLE P1505A 1.8 POLE Q1507R 3.6 POLE R1508C 2.7 POLE R1508C 5.4 POLE R1509G 5.4 POLE R1509S 31.5 POLE A1510V 433 POLE S1511F 113.5 POLE V1514L 11.7 POLE L1515P 7.2 POLE 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155.9 POLE R1566G 3.6 POLE A1567V 433 POLE A1567S 9.9 POLE A1567T 10.8 POLE I1568F 4.5 POLE R1570Q 0 POLE R1570Q 51.4 POLE L1573F 29.7 POLE A1574T 7.2 POLE A1574T 6.3 POLE A1574T 30.6 POLE Y1575C 36.9 POLE Y1575C 13.5 POLE K1576N 4.5 POLE K1576R 3.6 POLE E1577K 0.9 POLE E1577D 0 POLE E1577G 11.7 POLE R1579G 3.6 POLE R1579C 226.1 POLE R1579C 8.1 POLE R1580Q 45.9 POLE R1580Q 9 POLE G1581W 23.4 POLE G1581V 16.2 POLE G1581W 18.9 POLE P1582S 9.9 POLE L1584R 57.7 POLE L1584V 9.9 POLE I1585N 7.2 POLE A1586V 9 POLE A1586T 87.4 POLE V1587F 1.8 POLE V1587F 3.6 POLE A1597T 5.4 POLE S1598N 5.4 POLE S1598I 9.9 POLE I1600T 4.5 POLE P1601R 25.2 POLE P1601S 91.9 POLE V1602L 1.8 POLE E1604* 8.1 POLE E1604D 5.4 POLE E1604K 5.4 POLE P1607S 78.4 POLE I1611V 25.2 POLE V1613M 37.8 POLE D1615N 3.6 POLE K1616E 7.2 POLE I1617P 1.8 POLE N1618D 5.4 POLE G1620R 14.4 POLE V1621F 73 POLE V1621I 402.7 POLE V1621F 9 POLE W1624* 4.5 POLE W1624* 6.3 POLE W1624* 3.6 POLE W1624* 26.1 POLE R1626H 23.4 POLE R1626C 34.2 POLE R1626L 6.3 POLE R1626C 126.1 POLE R1630Q 1.8 POLE R1630W 0.9 POLE R1630Q 3.6 POLE R1630W 70.3 POLE R1630Q 11.7 POLE R1630W 0.9 POLE R1631C 3.6 POLE R1631C 0 POLE R1631L 1.8 POLE R1631H 62.2 POLE R1631H 3.6 POLE R1631C 56.8 POLE R1631C 40.5 POLE M1632I 24.3 POLE M1632I 32.4 POLE I1633V 18 POLE I1633M 31.5 POLE R1634H 3.6 POLE R1634H 17.1 POLE R1634H 3.6 POLE R1634H 29.7 POLE R1634C 0 POLE R1634C 12.6 POLE R1634H 2.7 POLE R1634H 12.6 POLE H1635Q 8.1 POLE H1635R 11.7 POLE Y1636S 1.8 POLE L1637F 84.7 POLE L1637F 5.4 POLE L1639P 1.8 POLE D1640G 8.1 POLE C1642F 13.5 POLE L1643V 2.7 POLE S1644L 7.2 POLE S1644L 3.6 POLE S1644L 6.3 POLE F1647S 450.5 POLE E1648* 18 POLE M1649I 3.6 POLE M1649I 17.1 POLE M1649I 36 POLE R1651M 17.1 POLE R1651M 22.5 POLE Y1652C 48.6 POLE H1654L 164 POLE H1654Y 5.4 POLE I1655V 11.7 POLE P1656L 18.9 POLE E1662K 21.6 POLE D1663H 5.4 POLE S1665T 0 POLE S1665F 7.2 POLE T1666A 9 POLE F1667S 309.9 POLE S1669P 8.1 POLE L1671F 6.3 POLE L1671F 99.1 POLE L1671P 22.5 POLE 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POLE R1826Q 7.2 POLE R1826W 4.5 POLE L1831P 18 POLE A1836V 26.1 POLE A1836V 0 POLE L1837M 80.2 POLE R1839H 1.8 POLE R1839C 3.6 POLE R1839C 5.4 POLE R1839L 4.5 POLE R1839H 0.9 POLE R1839C 121.6 POLE R1839C 59.5 POLE T1840P 1.8 POLE L1841F 0.9 POLE M1844I 4.5 POLE M1844I 39.6 POLE M1845R 5.4 POLE K1846Q 9 POLE K1846T 7.2 POLE L1848F 2.7 POLE L1850P 10.8 POLE L1852F 699 POLE I1853T 0.9 POLE A1854T 20.7 POLE A1854T 2.7 POLE A1854T 4.5 POLE A1854T 0.9 POLE E1855* 16.2 POLE F1856L 233.3 POLE F1856L 9.9 POLE K1857E 2.7 POLE R1858H 126.1 POLE R1858C 9 POLE R1858C 25.2 POLE R1858H 279.3 POLE R1858H 60.4 POLE G1860R 12.6 POLE G1860R 8.1 POLE G1860R 3.6 POLE G1860R 11.7 POLE S1862L 6.3 POLE I1864T 0.9 POLE Y1865C 27.9 POLE N1869D 17.1 POLE N1869Y 18 POLE R1870H 6.3 POLE R1870C 6.3 POLE R1870H 11.7 POLE R1870H 34.2 POLE R1870C 27 POLE R1870C 3.6 POLE R1870H 18 POLE I1871V 29.7 POLE T1875S 35.1 POLE R1878H 18.9 POLE R1878H 0.9 POLE R1878H 0 POLE R1878H 1.8 POLE R1878H 42.3 POLE R1878C 1.8 POLE R1878C 100.9 POLE R1878H 5.4 POLE R1878H 0.9 POLE R1878H 28.8 POLE R1878C 154.1 POLE R1878H 4.5 POLE R1878H 20.7 POLE R1878C 9.9 POLE R1878C 62.2 POLE R1878H 7.2 POLE R1879C 590 POLE R1879C 386.5 POLE R1879C 1.8 POLE R1879C 18.9 POLE V1880L 28.8 POLE A1883T 335.1 POLE I1884V 76.6 POLE A1885T 171.2 POLE A1885T 3.6 POLE A1885T 3.6 POLE A1885T 30.6 POLE A1885T 3.6 POLE Y1889C 34.2 POLE I1890V 18 POLE I1890V 10.8 POLE T1891I 170.3 POLE S1892I 11.7 POLE S1893N 12.6 POLE I1894T 703.6 POLE K1897Q 0.9 POLE T1904I 6.3 POLE I1905V 2.7 POLE S1906Y 203.6 POLE S1906Y 373.9 POLE S1906Y 240.5 POLE F1907L 3.6 POLE S1908F 315.3 POLE S1908C 7.2 POLE R1909Q 2.7 POLE R1909Q 0.9 POLE R1909Q 0 POLE C1910* 91.9 POLE W1911L 5.4 POLE L1914F 521.6 POLE L1914I 172.1 POLE L1914V 12.6 POLE M1917K 13.5 POLE M1917V 44.1 POLE S1920P 1.8 POLE N1921S 5.4 POLE N1921K 170.3 POLE N1921I 5.4 POLE N1921S 0.9 POLE Y1922C 5.4 POLE Y1922N 92.8 POLE G1923D 47.7 POLE G1923D 5.4 POLE G1924R 55 POLE G1924R 1.8 POLE G1924R 3.6 POLE 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119.8 POLE R2027M 47.7 POLE R2027S 50.5 POLE R2028M 105.4 POLE R2028T 7.2 POLE R2028M 29.7 POLE G2029V 18.9 POLE G2029V 10.8 POLE A2030S 119.8 POLE A2030T 2.7 POLE S2031N 90.1 POLE S2031G 2.7 POLE Q2032* 40.5 POLE Q2032R 12.6 POLE L2033F 19.8 POLE L2033V 2.7 POLE S2034F 107.2 POLE E2036V 7.2 POLE E2036V 2.7 POLE E2036V 5.4 POLE E2036V 6.3 POLE E2036V 34.2 POLE G2039W 31.5 POLE A2040V 500 POLE A2040E 4.5 POLE A2040V 26.1 POLE A2040V 11.7 POLE A2040V 36 POLE A2040V 66.7 POLE L2044F 4.5 POLE L2044F 63.1 POLE P2045L 174.8 POLE P2045L 119.8 POLE P2045L 229.7 POLE P2045L 17.1 POLE P2045L 152.3 POLE G2046V 12.6 POLE G2046* 13.5 POLE G2046* 27.9 POLE T2049I 114.4 POLE F2050L 4.5 POLE F2050L 27 POLE S2051F 7.2 POLE Q2052R 64 POLE Y2054D 479.3 POLE A2056S 12.6 POLE A2056T 3.6 POLE N2057D 56.8 POLE E2058G 3.6 POLE E2058G 2.7 POLE L2O59F 96.4 POLE L2059R 17.1 POLE T2060I 4.5 POLE T2060I 1.8 POLE S2062I 15.3 POLE S2062R 42.3 POLE F2063L 20.7 POLE F2063L 636.9 POLE I2066V 8.1 POLE T2067I 1.8 POLE T2067A 62.2 POLE Q2068* 20.7 POLE K2069N 3.6 POLE Q2071* 18 POLE K2072N 33.3 POLE K2072N 2.7 POLE V2074A 54.1 POLE S2077F 261.3 POLE S2077F 9.9 POLE R2078W 278.4 POLE R2078W 117.1 POLE E2082K 5.4 POLE E2082K 64.9 POLE E2082K 19.8 POLE S2084L 5.4 POLE S2084L 13.5 POLE S2084L 31.5 POLE E2085* 48.6 POLE E2085K 809 POLE M2086T 4.5 POLE M2086L 46.8 POLE M2086T 7.2 POLE M2086I 20.7 POLE M2086I 5.4 POLE P2088L 74.8 POLE P2091S 4.5 POLE P2091S 3.6 POLE G2092C 9.9 POLE G2092S 8.1 POLE G2092S 49.5 POLE G2092S 3.6 POLE G2092S 5.4 POLE S2093F 9.9 POLE S2093F 0.9 POLE S2093F 3.6 POLE H2094Y 17.1 POLE L2096V 2.7 POLE L2096V 10.8 POLE L2096V 3.6 POLE L2097R 1.8 POLE N2098D 45 POLE A2101D 5.4 POLE F2104L 118 POLE K2106E 156.8 POLE K2106R 0.9 POLE K2106R 3.6 POLE K2106R 6.3 POLE V2108L 9 POLE V2108A 4.5 POLE V2108A 12.6 POLE C2109W 14.4 POLE C2109R 1.8 POLE V2111M 17.1 POLE L2112V 0.9 POLE L2112V 18 POLE L2112V 1.8 POLE L2112P 1.8 POLE S2113C 18 POLE D2115E 112.6 POLE I2118M 69.4 POLE T2119P 64 POLE V2122M 22.5 POLE R2127P 3.6 POLE R2127* 2.7 POLE D2128N 45.9 POLE R2131H 15.3 POLE R2131C 0.9 POLE R2131H 7.2 POLE R2131C 11.7 POLE R2131L 29.7 POLE R2131H 58.6 POLE R2131H 1.8 POLE V2133L 49.5 POLE V2135D 15.3 POLE G2136S 18 POLE E2137Q 8.1 POLE E2137K 0.9 POLE E2137D 19.8 POLE E2137K 6.3 POLE E2137K 155 POLE F2138V 7.2 POLE S2139F 130.6 POLE S2139F 156.8 POLE E2140Q 5.4 POLE E2141Q 57.7 POLE A2142S 4.5 POLE F2144L 5.4 POLE R2145Q 9 POLE R2145* 54.1 POLE R2145Q 27.9 POLE R2145* 1.8 POLE R2145Q 27.9 POLE R2145* 15.3 POLE P2147S 2.7 POLE P2147S 8.1 POLE S2150F 3.6 POLE S2150F 81.1 POLE S2150F 38.7 POLE Y2151C 23.4 POLE Y2151N 9.9 POLE Y2151C 10.8 POLE Y2151C 4.5 POLE P2154T 699 POLE E2155K 13.5 POLE E2155K 27 POLE V2156I 6.3 POLE V2156I 5.4 POLE I2157N 93.7 POLE I2157V 4.5 POLE R2159H 18.9 POLE R2159L 11.7 POLE R2159H 50.5 POLE S2160C 7.2 POLE S2160I 7.2 POLE C2161S 2.7 POLE F2163I 2.7 POLE C2164F 25.2 POLE R2165C 4.5 POLE R2165C 0.9 POLE R2165C 2.7 POLE R2165C 35.1 POLE R2165C 1.8 POLE R2165C 6.3 POLE D2166N 9 POLE D2166Y 5.4 POLE D2166Y 14.4 POLE C2170R 3.6 POLE C2170R 28.8 POLE C2170R 27.9 POLE D2172H 9.9 POLE S2173C 91 POLE S2174F 83.8 POLE S2174F 12.6 POLE S2174F 12.6 POLE S2174F 142.3 POLE F2175V 122.5 POLE E2177Q 9 POLE E2177Q 9 POLE G2179A 0 POLE A2180V 51.4 POLE A2180V 46.8 POLE A2180V 230.6 POLE A2180V 3.6 POLE A2180V 450.5 POLE A2180V 44.1 POLE A2180V 176.6 POLE A2180V 10.8 POLE A2180V 65.8 POLE A2180V 13.5 POLE A2180V 6.3 POLE A2180V 2.7 POLE V2181E 4.5 POLE V2181F 6.3 POLE P2183F 93.7 POLE Q2184R 29.7 POLE W2185R 4.5 POLE W2185* 122.5 POLE L2186F 68.5 POLE Q2191H 21.6 POLE A2192V 2.7 POLE A2192V 7.2 POLE A2192V 71.2 POLE A2192V 1.8 POLE A2192V 2.7 POLE Y2194* 3.6 POLE D2195N 1.8 POLE D2195H 30.6 POLE D2195N 10.8 POLE D2195N 22.5 POLE D2195N 5.4 POLE D2195N 34.2 POLE S2197Y 2.7 POLE S2197F 18.9 POLE S2197F 3.6 POLE E2200D 22.5 POLE E2200* 20.7 POLE E2200K 15.3 POLE M2201L 9.9 POLE M2201V 6.3 POLE T2202M 2.7 POLE T2202M 0.9 POLE T2202M 9 POLE T2202M 2.7 POLE T2202M 7.2 POLE L2207V 4.5 POLE M2212V 45 POLE T2215S 9.9 POLE T2215I 48.6 POLE Q2217* 131.5 POLE Q2217* 25.2 POLE D2218N 36.9 POLE V2220I 7.2 POLE C2221S 17.1 POLE C2221R 14.4 POLE K2223R 2.7 POLE K2223R 3.6 POLE K2223R 20.7 POLE K2223R 1.8 POLE K2223R 1.8 POLE K2223R 3.6 POLE K2223R 1.8 POLE K2223R 3.6 POLE K2223R 6.3 POLE K2223R 1.8 POLE R2225H 1.8 POLE R2225G 45 POLE R2225C 5.4 POLE R2225C 12.6 POLE G2226W 40.5 POLE G2226W 16.2 POLE G2226R 53.2 POLE G2226A 9.9 POLE G2226R 0 POLE G2226R 14.4 POLE G2226R 1.8 POLE G2226R 9 POLE E2229D 4.5 POLE E2229K 100.9 POLE S2231G 33.3 POLE S2231N 126.1 POLE P2233L 182 POLE P2233S 20.7 POLE Y2235* 455.9 POLE C2238Y 2.7 POLE C2238W 46.8 POLE A2239T 0.9 POLE A2239T 9 POLE A2239T 46.8 POLE G2240R 25.2 POLE D2241H 2.7 POLE F2242L 5.4 POLE A2243T 7.2 POLE A2243T 3.6 POLE T2245S 3.6 POLE T2245S 4.5 POLE I2246M 2.7 POLE I2246T 1.8 POLE I2246V 1.8 POLE Q2249L 18 POLE Q2249* 71.2 POLE F2251L 6.3 POLE F2251L 6.3 POLE F2251L 15.3 POLE F2251L 3.6 POLE F2251L 20.7 POLE M2252V 3.6 POLE M2252L 12.6 POLE M2252V 1.8 POLE E2253Q 5.4 POLE E2253Q 5.4 POLE E2253Q 21.6 POLE Q2254H 28.8 POLE Q2254R 1.8 POLE I2255V 1.8 POLE G2256A 3.6 POLE G2256A 2.7 POLE G2256A 5.4 POLE R2259W 72.1 POLE R2259Q 2.7 POLE R2259Q 42.3 POLE R2259W 44.1 POLE Q2263* 10.8 POLE Q2263* 8.1 POLE Q2263* 3.6 POLE G2266F 15.3 POLE G2266S 18 POLE M2267I 0 POLE M2267I 31.5 POLE S2268L 333.3 POLE S2268* 2.7 POLE S2268L 7.2 POLE S2268L 5.4 POLE E2272* 5.4 POLE E2272K 31.5 POLE E2275K 561.3 POLE E2275D 36 POLE W2276L 36 POLE W2276C 10.8 POLE W2276C 1.8 POLE L2278P 8.1 POLE Q2279H 1.8 POLE K2280N 24.3 POLE N2281K 2.7 POLE P2282T 9 POLE Q2283* 1.8 POLE G2285C 2.7 POLE G2285S 1.8 POLE *2287Y 5.4 POLE Q2217fs*8 59.5 POLE Y1398fs*1 22.5 POLE E36fs*18 7.2 POLE L1766_E1767insEDMM 12.6 POLE L2112fs*23 8.1 POLE A706fs*86 6.3 POLE F699fs*11 36 POLE splice site 3459 + 1G > T 29.7 POLE K778fs*12 7.2 POLE splice site 726_801 + 128del204 9.9 POLE splice site 1021 − 1G > T 34.2 POLE L1983fs*16 9 POLE splice site 4290 + 1G > A 2.7 POLE S752del 40.5 POLE E1947_D1948insDEQENED 0.9 POLE splice site 6137 − 20_6165 > GAGTGA 3.6 POLE K495fs*11 7.2 POLE F699fs*11 22.5 POLE Y1398fs*53 4.5 POLE splice site 5552 + 1G > T 7.2 POLE E1707fs*4 4.5 POLE I230fs*6 0.9 POLE splice site 910 − 1G > A 179.3 POLE S80fs*10 13.5 POLE A1946fs*4 24.3 POLE splice site 5378 + 1G > T 3.6 POLE Y1398fs*1 9 POLE A840fs*10 18 POLE K1550fs*12 9.9 POLE N2260fs*28+ 2.7 POLE splice site 2561 + 1G > A 27.9 POLE Q125_G126 > HC 17.1 POLE I1144del 8.1 POLE H1680fs*81 18 POLE G302fs*42 4.5 POLE E396fs*16 56.8 POLE splice site 3795 + 1G > A 3.6 POLE splice site 802 − 2A > T 12.6 POLE V2234fs*54+ 17.1 POLE Q453fs*19 76.6 POLE E1202fs*10 5.4 POLE Q1152fs*3 31.5 POLE splice site 2026 + 1_2026 + 45del45 5.4 POLE splice site 5174 − 2A > C 11.7 POLE splice site 5174 − 2A > T 21.6 POLE G542fs*21 56.8 POLE E740fs*52 13.5 POLE T1891del 2.7 POLE V1725fs*36 14.4 POLE splice site 4445 − 1G > A 16.2 POLE N751fs*41 42.3 POLE L698fs*94 8.1 POLE F699fs*11 1.8 POLE F675fs*117 31.5 POLE splice site 3266_3275 + 16del26 9.9 POLE splice site 6658 − 2A > G 8.1 POLE Q1494fs*50 6.3 POLE splice site 62 + 1G > T 17.1 POLE V1446fs*3 30.6 POLE V1446fs*3 42.3 POLE splice site 5379 − 7_5382delCTTTCAGGATC 6.3 POLE splice site 4291 − 1G > T 17.1 POLE splice site 4499_4551 + 93del146 7.2 POLE K175del 39.6 POLE V1447fs*7 33.3 POLE splice site 3795 + 1G > A 114.4 POLE V1089fs*35 6.3 POLE A2030fs*29 7.2 POLE V574fs*1 8.1 POLE splice site 6137 − 1G > C 12.6 POLE A939fs*10 3.6 POLE splice site 3460 − 2A > G 14.4 POLE V2025fs*1 8.1 POLE L999fs*13 17.1 POLE K1550fs*12 58.6 POLE V82_D83 > VSTA*AVVLSA*AVVLLRQ 9.9 POLE D490fs*12 2.7 POLE K1276del 40.5 POLE V2220fs*69+ 27 POLE splice site 6331 − 41_6337del48 4.5 POLE V2025fs*1 9 POLE K1181del 2.7 POLE C501fs*1 6.3 POLE splice site 2026 + 1_2026 + 45del45 2.7 POLE M900_V901del 1.8 POLE D1211fs*1 4.5 POLE V1518fs*27 15.3 POLE splice site 3276 − 1G > C 0.9 POLE P135fs*68 72.1 POLE L698fs*94 5.4 POLE splice site 6331 − 2A > G 57.7 POLE D2218fs*3 2.7 POLE P269fs*26 7.2 POLE splice site 4006 − 1G > C 14.4 POLE G904fs*37 15.3 POLE R762fs*29 4.5 POLE L698fs*94 109 POLE D490fs*12 2.7 POLE K2228del 0 POLE splice site 801_801 + 1delTG 31.5 POLE A2030fs*18 56.8 POLE E137fs*12 2.7 POLE F699fs*11 27 POLE splice site 3060 + 1G > A 9 POLE P1223_A1224insA 0.9 POLE S1139fs*15 7.2 POLE L698fs*94 64.9 POLE splice site 3266_3275 + 16del26 4.5 POLE E2036_A2037del 26.1 POLE K1181del 3.6 POLE E740fs*51 5.4 POLE splice site 424 − 1G > A 3.6 POLE S871fs*38 2.7 POLE A2040fs*19 18.9 POLE splice site 1795 − 1G > T 96.4 POLE K175del 24.3 POLE R1364fs*5 69.4 POLE K1550fs*12 9.9 POLE S30fs*24 7.2 POLE splice site 2468 + 1G > T 14.4 POLE L1831fs*3 1.8 POLE E396fs*28 27 POLE splice site 1795 − 2A > T 28.8 POLE V1446fs*3 222.5 POLE P1223_A1224insA 5.4 POLE splice site 331 − 1G > A 2.7 POLE C600fs*1 8.1 POLE L1171fs*6 153.2 POLE splice site 1923 + 1G > T 27.9 POLE L698fs*94 59.5 POLE V1518fs*44 18 POLE splice site 3582 + 1G > T 19.8 POLE F699fs*11 52.3 POLE E521fs*7 6.3 POLE K1050fs*25 9.9 POLE K1174fs*45 9.9 POLE splice site 4290 + 1G > T 18.9 POLE P1223_A1224insA 2.7 POLE G1927fs*70 76.6 POLE splice site 1021 − 21_1030 > ACTGTCTGCTCTTTCTCTTCTTCAAGCTAT 33.3 POLE N1790fs*1 3.6 POLE R222fs*14 5.4 POLE S2173fs*115+ 7.2 POLE N1369fs*6 6.3 POLE splice site 1473 + 2T > C 45 POLE E396fs*16 22.5 POLE L1848fs*56 2.7 POLE V2025fs*34 41.4 POLE R1308fs*53 95.5 POLE G1542fs*20 14.4 POLE L46fs*6 18 POLE splice site 3266_3275 + 16del26 7.2 POLE C1788fs*8 6.3 POLE splice site 2026 + 1_2026 + 45del45 3.6 POLE D339fs*2 35.1 POLE G1262fs*99 18.9 POLE L698fs*94 39.6 POLE D316fs*28 2.7 POLE K1550fs*12 368.5 POLE splice site 6651_6657 + 6 > TGC 2.7 POLE L1850fs*54 2.7 POLE K1170fs*49 42.3 POLE N1843del 4.5 POLE T26fs*18 1.8 POLE V1446fs*3 46.8 POLE G24fs*30 132.4 POLE E1237fs*124 79.3 POLE K1550fs*12 36 POLE splice site 3266_3275 + 16del26 1.8 POLE splice site 4006 − 1G > C 25.2 POLE S5fs*49 7.2 POLE L1986fs*13 45 POLE F699fs*11 15.3 POLE splice site 1686 + 1G > T 19.8 POLE splice site 4149 + 1G > A 4.5 POLE F699fs*11 123.4 POLE K175del 2.7 POLE K2228del 2.7 POLE L493fs*13 4.5 POLE I1414fs*38 17.1 POLE L1295_V1301del 35.1 POLE E2229del 8.1 POLE splice site 4953 − 2A > G 10.8 POLE L698fs*94 38.7 POLE splice site 3460 − 1G > A 264 POLE F2175fs*26 1.8 POLE V1446fs*3 2.7 POLE splice site 4552 − 2_4552 − 1AG > TT 36 POLE I2157fs*45 5.4 POLE splice site 3266_3275 + 16del26 12.6 POLE K1276del 55.9 POLE K1550fs*12 32.4 POLE splice site 1106 + 1G > T 9 POLE L698fs*94 79.3 POLE F931fs*10 18 POLE splice site 6005 − 1G > A 87.4 POLE D1954_D1955insDEEERDG 6.3 POLE splice site 3266_3275 + 16del26 103.6 POLE splice site 3271 + 99_3275del104 5.4 POLE splice site 3253_3275 + 2del25 2.7 POLE V783fs*9 2.7 POLE S2173fs*115+ 18 POLE V1725fs*36 4.5 POLE splice site 4729 − 2A > T 106.3 POLE N1396fs*55 5.4 POLE I344fs*26 1.8 POLE splice site 2026 + 1_2026 + 45del45 2.7 POLE splice site 4006 − 1G > T 6.3 POLE splice site 5173 + 1G > T 16.2 POLE E903fs*38 26.1 POLE P1223_A1224insA 5.4 POLE splice site 6137 − 1_6137GG > AA 23.4 POLE splice site 3379 − 2A > G 9 POLE splice site 286 − 1G > A 6.3 POLE splice site 6531 + 1G > A 18 POLE C1788fs*8 1.8 POLE P1223_A1224insA 67.6 POLE W243fs*20 30.6 POLE S2197fs*91+ 159.5 POLE splice site 2561 + 1G > T 16.2 POLE Q2071fs*52 1.8 POLE splice site 1687 − 1G > C 19.8 POLE splice site 2864 + 1G > A 9.9 POLE L120fs*82 4.5 POLE splice site 5378 + 1G > T 7.2 POLE D319fs*3 21.6 POLE R10fs*16 0.9 POLE splice site 3266_3275 + 16del27 2.7 POLE splice site 6747 + 1G > T 9.9 POLE splice site 3266_3275 + 16del26 8.1 POLE I1326fs*58 2.7 POLE splice site 4291 − 1G > T 1.8 POLE splice site 2173 + 2T > A 13.5 POLE V1863_I1864insI 9 POLE Q2254fs*28+ 10.8 POLE F699fs*11 1.8 POLE splice site 5553 − 1G > A 34.2 POLE F1708fs*53 80.2 POLE splice site 6658 − 2A > G 3.6 POLE F699fs*11 68.5 POLE V1887fs*36 12.6 POLE splice site 6658 − 2A > G 21.6 POLE K1550fs*11 54.1 POLE I128fs*21 23.4 POLD1 Gene AA change Mutation burden POLD1 M1L 4.5 POLD1 M1T 17.1 POLD1 D2H 4.5 POLD1 G3S 9 POLD1 R6W 48.6 POLD1 R6W 6.3 POLD1 R6W 7.2 POLD1 P7S 23.4 POLD1 P7S 64 POLD1 P9L 69.4 POLD1 P9S 64 POLD1 P9S 104.5 POLD1 G10E 73.9 POLD1 P11H 230.6 POLD1 G12R 0 POLD1 G12R 2.7 POLD1 G12W 41.4 POLD1 G12V 87.4 POLD1 V13L 6.3 POLD1 V13L 2.7 POLD1 V13L 25.2 POLD1 V13M 1.8 POLD1 V13M 6.3 POLD1 P14S 15.3 POLD1 K16E 4.5 POLD1 K16E 4.5 POLD1 R17L 7.2 POLD1 A18T 76.6 POLD1 A18S 15.3 POLD1 R19L 49.5 POLD1 R19L 9.9 POLD1 G20R 38.7 POLD1 G21S 96.4 POLD1 G21S 8.1 POLD1 G21C 2.7 POLD1 G21V 18.9 POLD1 G21N 47.7 POLD1 G21V 9.9 POLD1 G21C 12.6 POLD1 L22F 0.9 POLD1 L22P 2.7 POLD1 D24N 9 POLD1 D27N 26.1 POLD1 D27G 4.5 POLD1 A28T 31.5 POLD1 P29H 91.9 POLD1 R30L 31.5 POLD1 R30L 10.8 POLD1 P31Q 165.8 POLD1 F34L 0.9 POLD1 F34I 67.6 POLD1 F34L 208.1 POLD1 E35K 2.7 POLD1 E35K 0.9 POLD1 E35K 4.5 POLD1 D37N 4.5 POLD1 D37N 1.8 POLD1 D37N 2.7 POLD1 D37N 29.7 POLD1 D37N 9 POLD1 A39T 44.1 POLD1 A39T 114.4 POLD1 M41L 2.7 POLD1 E42* 25.2 POLD1 M44V 5.4 POLD1 M44V 42.3 POLD1 M44I 20.7 POLD1 E47K 4.5 POLD1 H48Y 668.5 POLD1 R49S 8.1 POLD1 E52K 30.6 POLD1 Q53L 10.8 POLD1 E54V 24.3 POLD1 E57K 75.7 POLD1 E57K 48.6 POLD1 L58V 72.1 POLD1 Q59H 28.8 POLD1 Q59H 4.5 POLD1 S60P 15.3 POLD1 S60L 4.5 POLD1 S60P 48.6 POLD1 E63* 19.8 POLD1 E63K 1.8 POLD1 E63K 3.6 POLD1 E63K 0.9 POLD1 E63K 3.6 POLD1 V65L 2.7 POLD1 D67N 4.5 POLD1 D67N 13.5 POLD1 G68W 33.3 POLD1 G68A 23.4 POLD1 G68V 16.2 POLD1 G68W 18 POLD1 G68R 27.9 POLD1 G68R 5.4 POLD1 G68R 0.9 POLD1 P71S 122.5 POLD1 S73L 19.8 POLD1 P77F 31.5 POLD1 P77L 121.6 POLD1 R78C 26.1 POLD1 R78C 5.4 POLD1 R78C 3.6 POLD1 R78H 10.8 POLD1 R78C 15.3 POLD1 R78C 2.7 POLD1 R81Q 107.2 POLD1 R81W 3.6 POLD1 R81Q 15.3 POLD1 R81Q 55 POLD1 R81P 6.3 POLD1 T83I 9 POLD1 A86P 2.7 POLD1 A86V 2.7 POLD1 A86G 6.3 POLD1 A86V 24.3 POLD1 A86V 2.7 POLD1 A86S 0.9 POLD1 A86V 753.2 POLD1 L87P 0.9 POLD1 P89S 27.9 POLD1 Q90R 2.7 POLD1 Q90L 4.5 POLD1 E92* 6.3 POLD1 E92Q 13.5 POLD1 E92K 368.5 POLD1 L94F 83.8 POLD1 I95F 12.6 POLD1 I95V 153.2 POLD1 Q97* 4.5 POLD1 E100* 28.8 POLD1 D102Y 45 POLD1 H103Q 94.6 POLD1 Y104C 6.3 POLD1 V105A 6.3 POLD1 V105M 1.8 POLD1 G106C 30.6 POLD1 A108V 55.9 POLD1 A108S 84.7 POLD1 A108V 0 POLD1 A108V 24.3 POLD1 A108V 493.7 POLD1 Q109* 8.1 POLD1 Q109E 2.7 POLD1 P110R 7.2 POLD1 P110H 59.5 POLD1 V111L 10.8 POLD1 V111A 0.9 POLD1 P112H 541.36 POLD1 P112H 63.1 POLD1 G113V 52.3 POLD1 G114V 48.6 POLD1 G114W 6.3 POLD1 R119C 10.8 POLD1 R119C 12.6 POLD1 R119C 9.9 POLD1 R119C 122.5 POLD1 R119C 3.6 POLD1 R119C 1.8 POLD1 R119C 2.7 POLD1 R119C 37.8 POLD1 R119C 3.6 POLD1 R119C 2.7 POLD1 G120R 1.8 POLD1 G120S 4.5 POLD1 G120S 4.5 POLD1 G120N 48.6 POLD1 G120R 12.6 POLD1 G120V 20.7 POLD1 S121F 74.8 POLD1 S121F 22.5 POLD1 S121F 98.2 POLD1 S121F 4.5 POLD1 S121F 27 POLD1 S121F 36 POLD1 V122M 5.4 POLD1 V122M 4.5 POLD1 P123H 118 POLD1 P123L 521.6 POLD1 P123H 31.5 POLD1 R126C 55.9 POLD1 R126C 5.4 POLD1 R126L 28.8 POLD1 R126C 101.8 POLD1 R126H 5.4 POLD1 R126H 73 POLD1 R126C 6.3 POLD1 R126C 64 POLD1 R126H 19.8 POLD1 R126H 76.6 POLD1 A127T 2.7 POLD1 A127V 32.4 POLD1 F128S 587 POLD1 G129R 55.9 POLD1 G129R 6.3 POLD1 V130I 3.6 POLD1 V130I 455.9 POLD1 D132N 3.6 POLD1 D132N 305.4 POLD1 D132Y 20.7 POLD1 D132Y 15.3 POLD1 S136F 0.9 POLD1 S136A 455.9 POLD1 S136F 215.3 POLD1 V137F 8.1 POLD1 C138Y 170.3 POLD1 C138Y 72.1 POLD1 C139* 6.3 POLD1 G143C 19.8 POLD1 G143V 8.1 POLD1 G143S 4.5 POLD1 G143S 94.6 POLD1 G143S 65.8 POLD1 G143S 0.9 POLD1 G143C 37.8 POLD1 G143S 2.7 POLD1 P146L 24.3 POLD1 P146S 2.7 POLD1 P151L 214.4 POLD1 P151L 458.6 POLD1 A152S 27 POLD1 P153S 27 POLD1 P153L 1.8 POLD1 P153L 7.2 POLD1 P154S 37.8 POLD1 P154S 155 POLD1 P154S 47.7 POLD1 P154R 8.1 POLD1 G155A 5.4 POLD1 P158T 11.7 POLD1 P158S 0.9 POLD1 E159K 39.6 POLD1 E159K 18 POLD1 E159K 95.5 POLD1 E159K 1.8 POLD1 E159K 14.4 POLD1 E159K 8.1 POLD1 E159K 3.6 POLD1 H160Y 7.2 POLD1 H160Y 5.4 POLD1 H160Q 7.2 POLD1 D163Y 7.2 POLD1 R166W 3.6 POLD1 R166W 20.7 POLD1 R166W 4.5 POLD1 R166Q 0 POLD1 R166W 0.9 POLD1 R166W 4.5 POLD1 R166Q 7.2 POLD1 R166W 18 POLD1 R166Q 6.3 POLD1 R166W 3.6 POLD1 L170F 455.9 POLD1 L170S 59.5 POLD1 I172M 21.6 POLD1 R174L 9.9 POLD1 R174W 72.1 POLD1 R174W 1.8 POLD1 R174W 10.8 POLD1 R174L 6.3 POLD1 D175F 11.7 POLD1 S176R 9 POLD1 R177H 25.2 POLD1 R177H 11.7 POLD1 R177C 301.8 POLD1 G178W 5.4 POLD1 G178W 25.2 POLD1 G178V 18.9 POLD1 G178R 20.7 POLD1 G178R 1.8 POLD1 G178R 2.7 POLD1 G178V 119.8 POLD1 G178R 2.7 POLD1 G178R 2.7 POLD1 G178R 2.7 POLD1 L182M 0 POLD1 G184R 4.5 POLD1 G184E 178.4 POLD1 G184R 75.7 POLD1 G184V 51.4 POLD1 G184W 50.5 POLD1 A186V 133.3 POLD1 A186V 1.8 POLD1 V187M 0 POLD1 V187L 9.9 POLD1 V187G 7.2 POLD1 L188R 3.6 POLD1 L188R 2.7 POLD1 L188R 3.6 POLD1 L188R 4.5 POLD1 L188R 1.8 POLD1 A189S 28.8 POLD1 A189S 4.5 POLD1 A189D 51.4 POLD1 A189S 6.3 POLD1 V190L 19.8 POLD1 R195L 15.3 POLD1 E196K 65.8 POLD1 M198L 36.9 POLD1 M198V 9.9 POLD1 Y201H 0.9 POLD1 Y201H 21.6 POLD1 H202N 44.1 POLD1 H202Q 0.9 POLD1 H202Q 12.6 POLD1 G203V 12.6 POLD1 G203R 0.9 POLD1 G203W 9 POLD1 G203V 34.2 POLD1 G203R 105.4 POLD1 G203R 4.5 POLD1 G203R 18.9 POLD1 G203R 1.8 POLD1 H204Q 79.3 POLD1 G205S 112.6 POLD1 G205S 7.2 POLD1 G205C 8.1 POLD1 G205S 6.3 POLD1 P206S 98.2 POLD1 S207F 100.9 POLD1 S207F 16.2 POLD1 S207F 104.5 POLD1 P208L 74.8 POLD1 P208L 38.7 POLD1 P208L 27.9 POLD1 P208S 445.9 POLD1 P208L 30.6 POLD1 P208R 89.2 POLD1 P208L 0 POLD1 P208S 9.9 POLD1 P208S 53.2 POLD1 L210M 94.6 POLD1 L210V 3.6 POLD1 R211C 14.4 POLD1 R211H 4.5 POLD1 R211C 578.4 POLD1 R211P 2.7 POLD1 R211H 4.5 POLD1 R211H 2.7 POLD1 R211H 10.8 POLD1 R211H 0.9 POLD1 R211H 5.4 POLD1 R211H 7.2 POLD1 R211H 35.1 POLD1 R211H 5.4 POLD1 R211C 9 POLD1 R211H 9 POLD1 R211H 12.6 POLD1 R211H 2.7 POLD1 R211L 4.5 POLD1 R211P 2.7 POLD1 V214M 2.7 POLD1 V214M 8.1 POLD1 V214M 11.7 POLD1 A215V 238.18 POLD1 A215V 0.9 POLD1 A215V 10.8 POLD1 A215V 9 POLD1 A215V 155 POLD1 A215V 7.2 POLD1 L216M 27 POLD1 R218L 22.5 POLD1 R218L 18.9 POLD1 V220L 22.5 POLD1 A221S 18.9 POLD1 A221T 108.1 POLD1 A221S 14.4 POLD1 A221S 0.9 POLD1 P222L 12.6 POLD1 P222S 445.9 POLD1 P222L 6.3 POLD1 P222L 52.3 POLD1 A223T 318.9 POLD1 R224H 37.8 POLD1 R224C 1.8 POLD1 R224C 12.6 POLD1 R224H 3.6 POLD1 R224C 13.5 POLD1 R224H 114.4 POLD1 R224H 11.7 POLD1 R224C 4.5 POLD1 R224C 10.8 POLD1 R224H 4.5 POLD1 R224C 58.6 POLD1 R224C 5.4 POLD1 R224C 1.8 POLD1 R224H 2.7 POLD1 R225H 9 POLD1 R225H 45 POLD1 R225H 10.8 POLD1 R225H 0.9 POLD1 R225P 71.2 POLD1 R225H 2.7 POLD1 R225C 18 POLD1 R225H 2.7 POLD1 R225H 8.1 POLD1 R225C 12.6 POLD1 R225H 3.6 POLD1 R225C 2.7 POLD1 L227M 665.8 POLD1 E228K 36 POLD1 E228Q 27.9 POLD1 Q229R 0 POLD1 Q229R 8.1 POLD1 Q229H 8.1 POLD1 G230D 14.4 POLD1 R232H 587 POLD1 R232H 64.9 POLD1 R232C 3.6 POLD1 R232C 650.5 POLD1 R232C 224.3 POLD1 R232C 108.1 POLD1 V233L 13.5 POLD1 G235S 6.3 POLD1 T238M 5.4 POLD1 T238M 3.6 POLD1 T238M 4.5 POLD1 T238M 2.7 POLD1 T238M 2.7 POLD1 T238M 5.4 POLD1 T238M 1.8 POLD1 A242T 43.2 POLD1 A242T 4.5 POLD1 A242V 3.6 POLD1 A242V 5.4 POLD1 A242T 56.8 POLD1 A242T 1.8 POLD1 A242V 159.5 POLD1 P243S 112.6 POLD1 Y244F 36 POLD1 E245K 76.6 POLD1 E245K 122.5 POLD1 E245K 85.6 POLD1 A246V 51.4 POLD1 A246T 0.9 POLD1 V248I 12.6 POLD1 D249Y 4.5 POLD1 D249N 68.5 POLD1 E251D 44.1 POLD1 E251Q 36 POLD1 R253Q 5.4 POLD1 R253L 6.3 POLD1 F254L 20.7 POLD1 F254L 4.5 POLD1 M255I 8.1 POLD1 D259N 65.8 POLD1 D259Y 9.9 POLD1 V261I 64.9 POLD1 V261I 3.6 POLD1 V261I 6.3 POLD1 V261F 1.8 POLD1 V261I 8.1 POLD1 C263Y 230.6 POLD1 C263Y 668.5 POLD1 C263Y 5.4 POLD1 N264D 18.9 POLD1 W265R 36.9 POLD1 E267Q 15.3 POLD1 P269S 103.6 POLD1 P269S 334.2 POLD1 G271R 5.4 POLD1 G271R 1.8 POLD1 A274T 60.4 POLD1 A274T 2.7 POLD1 A274S 10.8 POLD1 A274T 76.6 POLD1 A274T 0.9 POLD1 A274V 119.8 POLD1 L275V 37.8 POLD1 R276M 51.4 POLD1 R276S 5.4 POLD1 R276M 59.5 POLD1 K278N 0.9 POLD1 E279D 44.1 POLD1 A281V 21.6 POLD1 T282M 82.9 POLD1 T282M 2.7 POLD1 T282M 0.9 POLD1 T282M 41.4 POLD1 T282M 4.5 POLD1 T282M 159.5 POLD1 C284Y 7.2 POLD1 E287* 9.9 POLD1 E287Q 6.3 POLD1 A288T 7.2 POLD1 A288T 9 POLD1 A288V 116.2 POLD1 A288T 0 POLD1 D289N 68.5 POLD1 V290M 608.02 POLD1 V290M 2.7 POLD1 V290M 65.8 POLD1 V290M 0.9 POLD1 V290M 154.1 POLD1 V290L 19.8 POLD1 L291P 3.6 POLD1 W292L 20.7 POLD1 V295L 28.8 POLD1 V296G 12.6 POLD1 P299S 154.1 POLD1 P299S 159.5 POLD1 P300L 5.4 POLD1 P300L 226.1 POLD1 P300T 20.7 POLD1 P300L 5.4 POLD1 P300L 92.8 POLD1 E301K 18.9 POLD1 G302K 145.9 POLD1 P303S 74.8 POLD1 W304S 27 POLD1 W304M 33.3 POLD1 R306C 10.8 POLD1 R306H 4.5 POLD1 R306H 156.8 POLD1 R306H 17.1 POLD1 R306H 45 POLD1 R306H 19.8 POLD1 R306C 3.6 POLD1 R306C 31.5 POLD1 R306C 69.4 POLD1 A308V 4.5 POLD1 R311H 2.7 POLD1 R311C 314.4 POLD1 V312M 141.4 POLD1 V312M 0.9 POLD1 V312M 7.2 POLD1 V312M 15.3 POLD1 V312M 753.2 POLD1 V312M 0 POLD1 S314C 20.7 POLD1 D316N 2.7 POLD1 D316N 2.7 POLD1 E318K 182 POLD1 E318K 13.5 POLD1 E318A 19.8 POLD1 E318V 64.9 POLD1 C319Y 587 POLD1 C319Y 90.1 POLD1 A320S 12.6 POLD1 A320P 5.4 POLD1 A320T 15.3 POLD1 A320T 64 POLD1 A320S 36 POLD1 A320T 18 POLD1 G321C 36.9 POLD1 G321D 65.8 POLD1 R322H 9 POLD1 R322H 29.7 POLD1 R322C 1.8 POLD1 R322H 2.7 POLD1 R322C 38.7 POLD1 R322H 56.8 POLD1 R322H 4.5 POLD1 G324C 53.2 POLD1 G324D 15.3 POLD1 I325M 22.5 POLD1 I325V 1.8 POLD1 I325M 4.5 POLD1 P327S 170.3 POLD1 P327S 9 POLD1 P327R 10.8 POLD1 E328D 15.3 POLD1 E330Q 29.7 POLD1 E330K 3.6 POLD1 E330Q 12.6 POLD1 R331W 371.2 POLD1 R331Q 66.7 POLD1 R331P 16.2 POLD1 R331Q 0 POLD1 R331W 4.5 POLD1 R331Q 62.2 POLD1 R331Q 5.4 POLD1 R331W 753.2 POLD1 R331W 18.9 POLD1 R331W 2.7 POLD1 P333L 163.1 POLD1 P333S 7.2 POLD1 V334A 162.2 POLD1 Q336H 52.3 POLD1 Q336P 10.8 POLD1 S339L 1.8 POLD1 S339L 854.1 POLD1 S339L 98.2 POLD1 S339T 11.7 POLD1 G341D 5.4 POLD1 R343C 238.18 POLD1 R343C 2.7 POLD1 R343H 56.8 POLD1 R343H 4.5 POLD1 R343C 0 POLD1 R343H 14.4 POLD1 W344* 22.5 POLD1 E346Q 35.1 POLD1 E346K 155 POLD1 E346D 64.9 POLD1 P347L 0.9 POLD1 P347L 64.9 POLD1 P347L 0.9 POLD1 E348Q 2.7 POLD1 R352H 0 POLD1 R352H 9 POLD1 R352C 37.8 POLD1 R352H 5.4 POLD1 R352C 11.7 POLD1 R352H 9.9 POLD1 R352H 650.5 POLD1 R352H 4.5 POLD1 R352C 49.5 POLD1 R352C 31.5 POLD1 L353V 5.4 POLD1 T356I 6.3 POLD1 R358L 14.4 POLD1 R358Q 26.1 POLD1 R358Q 27.9 POLD1 P359H 60.4 POLD1 A361S 5.4 POLD1 A361V 26.1 POLD1 A361V 9 POLD1 A361V 6.3 POLD1 P362S 112.6 POLD1 P362S 98.2 POLD1 L364P 7.2 POLD1 L364M 55.9 POLD1 G365R 1.8 POLD1 G365S 20.7 POLD1 A366P 7.2 POLD1 Q369H 160.4 POLD1 S370N 1.8 POLD1 S370N 118.9 POLD1 E372K 104.5 POLD1 E372K 3.6 POLD1 E372K 1.8 POLD1 E372K 0.9 POLD1 K373E 5.4 POLD1 K373R 8.1 POLD1 E375K 122.5 POLD1 W381S 9 POLD1 S382F 100.9 POLD1 T383A 4.5 POLD1 F384I 2.7 POLD1 F384V 17.1 POLD1 R386H 4.5 POLD1 R386H 0 POLD1 I387N 51.4 POLD1 I387M 36.9 POLD1 M388I 100 POLD1 D389Y 49.5 POLD1 P390A 12.6 POLD1 P390A 17.1 POLD1 P390R 30.6 POLD1 D391Y 17.1 POLD1 D391E 14.4 POLD1 V392M 6.3 POLD1 V392L 37.8 POLD1 V392M 68.5 POLD1 V392M 2.7 POLD1 V392L 9 POLD1 V392M 5.4 POLD1 V392M 12.6 POLD1 V392M 2.7 POLD1 V392M 11.7 POLD1 V392M 0 POLD1 V392M 0 POLD1 I393V 19.8 POLD1 T394I 13.5 POLD1 G395S 4.5 POLD1 N397D 115.3 POLD1 I398M 5.4 POLD1 I398M 9 POLD1 I398M 13.5 POLD1 I398M 7.2 POLD1 D402N 52.3 POLD1 D402Y 17.1 POLD1 D402H 9.9 POLD1 D402N 26.1 POLD1 D402N 9.9 POLD1 D402N 7.2 POLD1 D402N 54.1 POLD1 D402N 315.3 POLD1 D402N 8.1 POLD1 L403I 67.6 POLD1 P404L 119.8 POLD1 L406P 311.7 POLD1 L406F 8.1 POLD1 S408F 11.7 POLD1 R409Q 4.5 POLD1 R409W 27.9 POLD1 R409L 8.1 POLD1 R409W 56.8 POLD1 R409Q 4.5 POLD1 Q411H 2.7 POLD1 T412I 55 POLD1 K414* 21.6 POLD1 V415I 320.7 POLD1 V415L 26.1 POLD1 P419S 103.6 POLD1 P419S 33.3 POLD1 P419S 62.2 POLD1 P419S 20.7 POLD1 G422D 7.2 POLD1 R423L 14.4 POLD1 R423H 35.1 POLD1 R423C 48.6 POLD1 R423H 13.5 POLD1 R423H 12.6 POLD1 V424L 8.1 POLD1 A425S 6.3 POLD1 A425S 2.7 POLD1 A425T 264 POLD1 A425S 1.8 POLD1 A425T 46.8 POLD1 G426S 0 POLD1 G426S 2.7 POLD1 G426S 12.6 POLD1 G426C 16.2 POLD1 G426S 59.5 POLD1 N430S 2.7 POLD1 N430S 0 POLD1 I431V 3.6 POLD1 I431V 2.7 POLD1 I431V 3.6 POLD1 I431V 0.9 POLD1 R432W 31.5 POLD1 R432W 0.9 POLD1 R432W 35.1 POLD1 R432W 7.2 POLD1 R432W 0.9 POLD1 R432W 31.5 POLD1 R432W 67.6 POLD1 R432W 7.2 POLD1 R432G 5.4 POLD1 R432W 4.5 POLD1 R432L 29.7 POLD1 R432W 27 POLD1 R432W 2.7 POLD1 S434A 0.9 POLD1 S434C 8.1 POLD1 S435L 38.7 POLD1 Q437R 35.1 POLD1 Q440* 10.8 POLD1 Q440H 1.8 POLD1 G442C 9 POLD1 R443Q 3.6 POLD1 R443W 67.6 POLD1 R444W 9 POLD1 R444Q 7.2 POLD1 R444Q 8.1 POLD1 R444W 1.8 POLD1 R444W 109 POLD1 K447M 175.7 POLD1 V449A 541.36 POLD1 V449I 180.2 POLD1 M451I 18.9 POLD1 V452L 15.3 POLD1 V452L 18.9 POLD1 V452L 41.4 POLD1 G453D 98.2 POLD1 R454L 31.5 POLD1 R454C 12.6 POLD1 R454C 23.4 POLD1 R454C 14.4 POLD1 R454C 34.2 POLD1 R454C 14.4 POLD1 V455L 25.2 POLD1 V455L 45.9 POLD1 Q456E 1.8 POLD1 Q456E 0.9 POLD1 Q456L 4.5 POLD1 D458G 590 POLD1 D458Y 25.2 POLD1 L460R 180.2 POLD1 V462G 5.4 POLD1 V462L 7.2 POLD1 L464Q 21.6 POLD1 R465L 15.3 POLD1 R465Q 114.4 POLD1 R465W 11.7 POLD1 R465Q 11.7 POLD1 E466K 39.6 POLD1 R470C 74.8 POLD1 R470H 4.5 POLD1 R470C 58.6 POLD1 R470H 39.6 POLD1 S471Y 590 POLD1 T473M 10.8 POLD1 T473M 4.5 POLD1 T473M 28.8 POLD1 T473A 25.2 POLD1 T473M 6.3 POLD1 N475S 1.8 POLD1 A476V 25.2 POLD1 A476S 53.2 POLD1 A476V 53.2 POLD1 V477M 0.9 POLD1 V477M 19.8 POLD1 V477M 42.3 POLD1 V477M 1.8 POLD1 S478I 6.3 POLD1 S478I 12.6 POLD1 F479S 1.8 POLD1 F479V 5.4 POLD1 F479S 29.7 POLD1 G483R 9 POLD1 E484K 170.3 POLD1 K486N 279.3 POLD1 E487Q 5.4 POLD1 E487Q 32.4 POLD1 E487V 1.8 POLD1 E487* 12.6 POLD1 V489M 0.9 POLD1 V489M 7.2 POLD1 V489M 4.5 POLD1 V489M 115.3 POLD1 V489L 62.2 POLD1 S492G 9 POLD1 I493M 7.2 POLD1 D496N 9.9 POLD1 Q498K 65.8 POLD1 Q498L 22.5 POLD1 G500R 2.7 POLD1 N501H 0 POLD1 D502H 15.3 POLD1 D502Y 8.1 POLD1 D502E 34.2 POLD1 Q503E 0.9 POLD1 R505C 5.4 POLD1 R505H 12.6 POLD1 R506C 9 POLD1 R506C 13.5 POLD1 R506P 10.8 POLD1 R506C 3.6 POLD1 R506C 8.1 POLD1 R507H 97.3 POLD1 R507C 1.8 POLD1 R507H 25.2 POLD1 A509P 12.6 POLD1 A509V 26.1 POLD1 V510L 10.8 POLD1 V510M 94.6 POLD1 C512R 2.7 POLD1 D515N 26.1 POLD1 L518M 23.4 POLD1 L518M 19.8 POLD1 L518M 16.2 POLD1 P519L 8.1 POLD1 L520P 32.4 POLD1 R521W 302 POLD1 R521W 17.1 POLD1 R525W 8.1 POLD1 R525Q 10.8 POLD1 R525Q 10.8 POLD1 R525W 44.1 POLD1 R525Q 4.5 POLD1 R525Q 2.7 POLD1 N531S 5.4 POLD1 A532T 608.02 POLD1 A532V 1.8 POLD1 A532V 142.3 POLD1 V533M 14.4 POLD1 V533M 9 POLD1 E534D 19.8 POLD1 E534D 3.6 POLD1 A536G 99.1 POLD1 A536V 455.9 POLD1 A536V 4.5 POLD1 G540C 3.6 POLD1 V541M 590 POLD1 V541L 8.1 POLD1 V541M 1.8 POLD1 V541M 0 POLD1 L546V 20.7 POLD1 L546M 49.5 POLD1 L547P 11.7 POLD1 L547F 386.5 POLD1 S548C 23.4 POLD1 S548I 4.5 POLD1 R549S 40.5 POLD1 R549C 5.4 POLD1 R549H 5.4 POLD1 R549H 2.7 POLD1 R549H 0 POLD1 R549C 171.2 POLD1 V556I 1.8 POLD1 V556I 6.3 POLD1 V556I 9.9 POLD1 V556I 2.7 POLD1 V556I 5.4 POLD1 V556I 18.9 POLD1 V556I 9 POLD1 V556I 17.1 POLD1 V556I 2.7 POLD1 V556A 45.9 POLD1 S557F 3.6 POLD1 S557F 64 POLD1 Q558R 109 POLD1 Q558* 3.6 POLD1 R561Q 56.8 POLD1 R561L 2.7 POLD1 R561Q 12.6 POLD1 R561Q 2.7 POLD1 Q562H 57.7 POLD1 A563V 21.6 POLD1 H565P 7.2 POLD1 H565P 7.2 POLD1 H565L 73 POLD1 L569P 12.6 POLD1 L569R 2.7 POLD1 P571L 112.6 POLD1 P571L 130.6 POLD1 V572M 1.8 POLD1 V572M 0.9 POLD1 V572M 50.5 POLD1 V573L 4.5 POLD1 S575L 10.8 POLD1 S575L 5.4 POLD1 G578S 32.4 POLD1 G578D 51.4 POLD1 G578C 10.8 POLD1 E579Q 7.2 POLD1 E579K 3.6 POLD1 E579K 4.5 POLD1 E579K 7.2 POLD1 D580G 81.98 POLD1 D580H 52.3 POLD1 T582M 7.2 POLD1 T582M 0 POLD1 T582M 0.9 POLD1 T582M 9.9 POLD1 G583R 22.5 POLD1 G583V 119.8 POLD1 G583V 8.1 POLD1 G583R 18 POLD1 V586F 1.8 POLD1 E588K 4.5 POLD1 G592R 33.3 POLD1 G592R 9 POLD1 Y593H 324.3 POLD1 V596I 42.3 POLD1 P597S 100.9 POLD1 P597S 13.5 POLD1 T600S 9.9 POLD1 F603L 23.4 POLD1 S604Y 4.5 POLD1 S604Y 0 POLD1 L606M 196 POLD1 L606M 295 POLD1 L606M 228.8 POLD1 L606M 51.4 POLD1 L606M 311.7 POLD1 L606V 3.6 POLD1 Y607C 68.5 POLD1 P608L 2.7 POLD1 M612T 1.8 POLD1 A613G 7.2 POLD1 A613V 315.3 POLD1 A613G 23.4 POLD1 L616M 2.7 POLD1 C617G 5.4 POLD1 T620M 57.7 POLD1 T620M 650.5 POLD1 T620M 44.1 POLD1 L622F 69.4 POLD1 R623Q 236.9 POLD1 R623L 39.6 POLD1 R623W 311.7 POLD1 R623W 12.6 POLD1 R623W 155 POLD1 R623W 4.5 POLD1 R623W 3.6 POLD1 R623W 3.6 POLD1 R623Q 8.1 POLD1 R623Q 80.2 POLD1 R623W 373.9 POLD1 R623W 215.3 POLD1 R623W 23.4 POLD1 R623W 5.4 POLD1 G625R 107.2 POLD1 T626I 3.6 POLD1 A627T 76.6 POLD1 A627T 235.1 POLD1 A627T 51.4 POLD1 K629I 9.9 POLD1 K629I 0.9 POLD1 G631D 8.1 POLD1 G631R 90.1 POLD1 L632M 141 POLD1 L632M 228 POLD1 L632M 296 POLD1 L632M 121 POLD1 E634K 9 POLD1 E634A 66.7 POLD1 E634K 9.9 POLD1 D635N 578.4 POLD1 F637L 170.3 POLD1 I638V 3.6 POLD1 R639K 45.9 POLD1 T640A 1.8 POLD1 T640I 122.5 POLD1 T640N 5.4 POLD1 T640N 0 POLD1 T640N 3.6 POLD1 P641L 25.2 POLD1 T642I 112.6 POLD1 G643W 83.8 POLD1 G643W 79.3 POLD1 G643L 7.2 POLD1 D644G 6.3 POLD1 D644N 2.7 POLD1 D644N 23.4 POLD1 D644N 9.9 POLD1 D644E 4.5 POLD1 D644N 26.1 POLD1 D644A 36 POLD1 E645D 33.3 POLD1 E645K 231.5 POLD1 E645K 3.6 POLD1 E645K 9 POLD1 F646C 4.5 POLD1 F646Y 14.4 POLD1 F646Y 9.9 POLD1 L648I 228 POLD1 K648N 6.3 POLD1 T649P 3.6 POLD1 T649P 8.1 POLD1 T649I 229.7 POLD1 T649P 8.1 POLD1 S650L 45 POLD1 V651L 49.5 POLD1 R652W 27.9 POLD1 R652W 47.7 POLD1 R652Q 36.9 POLD1 R652Q 13.5 POLD1 R652Q 5.4 POLD1 R652W 13.5 POLD1 R652W 447.8 POLD1 R652Q 3.6 POLD1 R652L 16.2 POLD1 R652Q 8.1 POLD1 K653N 65.8 POLD1 G654V 26.1 POLD1 G654L 12.6 POLD1 G654V 12.6 POLD1 L656M 56.8 POLD1 P657L 1.8 POLD1 N662D 8.1 POLD1 S665N 4.5 POLD1 S665N 23.4 POLD1 S665C 22.5 POLD1 R667L 14.4 POLD1 K671M 19.8 POLD1 A675V 5.4 POLD1 A675S 17.1 POLD1 E677Q 49.5 POLD1 D679G 56.8 POLD1 P680L 3.6 POLD1 P680L 9 POLD1 P680L 3.6 POLD1 P680A 29.7 POLD1 P680L 6.3 POLD1 L681V 0 POLD1 R682W 311.7 POLD1 R682W 46.8 POLD1 R682L 34.2 POLD1 R682Q 6.3 POLD1 R682L 18.9 POLD1 R683H 4.5 POLD1 R683C 64 POLD1 R683H 64 POLD1 V685F 9 POLD1 D687H 18.9 POLD1 D687Y 19.8 POLD1 D687Y 131.5 POLD1 G688* 27.9 POLD1 G688E 3.6 POLD1 R689W 259.5 POLD1 R689W 585.6 POLD1 R689W 578.4 POLD1 R689L 12.6 POLD1 R689W 447.8 POLD1 R689W 12.6 POLD1 R689P 6.3 POLD1 Q690* 12.6 POLD1 A692V 4.5 POLD1 A692S 4.5 POLD1 L693M 35.1 POLD1 V695M 4.5 POLD1 A697T 699 POLD1 N698I 57.7 POLD1 S699F 138.7 POLD1 S699F 118.9 POLD1 S699F 53.2 POLD1 S699F 159.5 POLD1 S699F 29.7 POLD1 S699F 156.8 POLD1 S699T 445.9 POLD1 S699F 98.2 POLD1 V700I 0.9 POLD1 V700I 0 POLD1 V700I 3.6 POLD1 V700I 80.2 POLD1 V700I 7.2 POLD1 T704A 9.9 POLD1 G705C 176.6 POLD1 G705S 20.7 POLD1 A706T 13.5 POLD1 A706T 52.3 POLD1 A706S 15.3 POLD1 G709C 3.6 POLD1 P712S 98.2 POLD1 C713G 230.6 POLD1 C713F 110.8 POLD1 C713W 48.6 POLD1 C713F 49.5 POLD1 E715K 15.3 POLD1 E715K 0.9 POLD1 E715D 72.1 POLD1 I716V 10.8 POLD1 S717A 230.6 POLD1 S717* 73.9 POLD1 L719Q 228 POLD1 V720I 17.1 POLD1 G724R 9.9 POLD1 R725C 2.7 POLD1 R725C 64.9 POLD1 R725C 145.9 POLD1 R725H 12.6 POLD1 R725H 2.7 POLD1 R725C 27 POLD1 Q726* 86.5 POLD1 I728M 107.2 POLD1 E729Q 2.7 POLD1 K730Q 82.9 POLD1 K730Q 10.8 POLD1 T731I 608.02 POLD1 Q733E 2.7 POLD1 V735M 114.4 POLD1 E736G 2.7 POLD1 S737C 5.4 POLD1 S737Y 301.8 POLD1 S737C 1.8 POLD1 T740A 2.7 POLD1 T740A 7.2 POLD1 V741M 3.6 POLD1 E742D 4.5 POLD1 E742G 0.9 POLD1 E742D 7.2 POLD1 E742Q 5.4 POLD1 G744V 20.7 POLD1 G744D 2.7 POLD1 S746G 57.7 POLD1 T747I 38.7 POLD1 S748G 29.7 POLD1 A749P 11.7 POLD1 A749T 13.5 POLD1 V751M 7.2 POLD1 Y753H 5.4 POLD1 G754S 75.7 POLD1 V759G 18 POLD1 R762P 11.7 POLD1 R762* 70.3 POLD1 R762* 2.7 POLD1 R762* 0.9 POLD1 R762* 8.1 POLD1 G764D 2.7 POLD1 V765M 4.5 POLD1 V765M 8.1 POLD1 V765M 1.8 POLD1 V765M 18.9 POLD1 S767L 1.8 POLD1 A769G 13.5 POLD1 E770D 8.1 POLD1 E770K 19.8 POLD1 E770K 11.7 POLD1 A771V 0.9 POLD1 L774V 6.3 POLD1 L774V 1.8 POLD1 G775R 52.3 POLD1 R776Q 25.2 POLD1 R776W 5.4 POLD1 R776W 1.8 POLD1 R776Q 107.2 POLD1 R776Q 5.4 POLD1 R776L 17.1 POLD1 R776W 7.2 POLD1 R776W 5.4 POLD1 R776W 27.9 POLD1 R776W 17.1 POLD1 E777K 4.5 POLD1 E777K 3.6 POLD1 A778V 3.6 POLD1 A778V 28.8 POLD1 A778V 159.5 POLD1 A778V 0 POLD1 A779T 50.5 POLD1 A779V 141.4 POLD1 A779V 46.8 POLD1 A779T 13.5 POLD1 D780N 25.2 POLD1 D780N 63.1 POLD1 D780H 13.5 POLD1 D780E 6.3 POLD1 W781* 156.8 POLD1 W781C 34.2 POLD1 W781L 3.6 POLD1 S783L 13.5 POLD1 S783T 5.4 POLD1 H785Y 45.9 POLD1 H785N 133.3 POLD1 P787L 5.4 POLD1 P787L 0.9 POLD1 P787L 7.2 POLD1 P787L 67.6 POLD1 P787L 17.1 POLD1 P787L 37.8 POLD1 S788L 4.5 POLD1 S788L 15.3 POLD1 S788L 0.9 POLD1 P789L 6.3 POLD1 R791L 20.7 POLD1 R791W 141.4 POLD1 R791W 152.3 POLD1 R791W 94.6 POLD1 E795Q 9.9 POLD1 E795Q 4.5 POLD1 E795D 30.6 POLD1 E795D 28.8 POLD1 E795K 27 POLD1 E795K 19.8 POLD1 E795Q 11.7 POLD1 Y798C 15.3 POLD1 F799L 2.7 POLD1 P800S 78.4 POLD1 I804M 14.4 POLD1 I804M 7.2 POLD1 S805I 25.2 POLD1 K806* 12.6 POLD1 R808H 123.4 POLD1 Y809* 9.9 POLD1 Y809* 6.3 POLD1 A810E 1.8 POLD1 A810S 13.5 POLD1 G811A 54.1 POLD1 G811N 97.3 POLD1 L812V 8.1 POLD1 F814L 2.7 POLD1 S815F 8.1 POLD1 S815F 100 POLD1 S815F 21.6 POLD1 S816C 8.1 POLD1 R817Q 0 POLD1 R817L 35.1 POLD1 P818L 9 POLD1 D819N 37.8 POLD1 D819N 1.8 POLD1 D819N 9.9 POLD1 A820S 39.6 POLD1 A820T 1.8 POLD1 A820T 9.9 POLD1 A820T 1.8 POLD1 A820T 9 POLD1 H821Q 2.7 POLD1 R823G 229.7 POLD1 R823L 18.9 POLD1 M824I 76.6 POLD1 C826Y 126.1 POLD1 L829P 76.6 POLD1 E830* 9.9 POLD1 E830* 6.3 POLD1 A831S 16.2 POLD1 V832L 9 POLD1 V832L 8.1 POLD1 R833L 33.3 POLD1 R833C 9.9 POLD1 R833L 9.9 POLD1 R833L 21.6 POLD1 R834M 4.5 POLD1 R834S 12.6 POLD1 D835N 8.1 POLD1 D835E 13.5 POLD1 C837R 56.8 POLD1 P838S 75.7 POLD1 P838S 182 POLD1 P838L 86.5 POLD1 P838S 41.4 POLD1 L839F 98.2 POLD1 L839V 4.5 POLD1 V840M 54.1 POLD1 N842K 41.4 POLD1 V844I 10.8 POLD1 A846V 12.6 POLD1 S847L 8.1 POLD1 R849C 4.5 POLD1 R849C 0.9 POLD1 R849C 0.9 POLD1 R849C 0.9 POLD1 R849C 0.9 POLD1 R849C 9.9 POLD1 R849C 20.7 POLD1 R850C 2.7 POLD1 L852P 20.7 POLD1 L852F 0.9 POLD1 D854H 4.5 POLD1 D854Y 45 POLD1 D854Y 1.8 POLD1 R855* 7.2 POLD1 R855L 7.2 POLD1 R855L 0.9 POLD1 R855Q 6.3 POLD1 R855G 76.6 POLD1 R855G 2.7 POLD1 R855L 5.4 POLD1 R855L 0.9 POLD1 D856H 9.9 POLD1 E858K 23.4 POLD1 E858D 9 POLD1 A860V 4.5 POLD1 A860V 854.1 POLD1 A860T 33.3 POLD1 A860V 105.4 POLD1 A860V 0.9 POLD1 A860V 38.7 POLD1 A862P 118.9 POLD1 H863Y 15.3 POLD1 A864P 9.9 POLD1 A864P 4.5 POLD1 A864T 16.2 POLD1 A864T 0.9 POLD1 A864S 17.1 POLD1 A864S 14.4 POLD1 D866E 4.5 POLD1 I868V 164.9 POLD1 S869L 4.5 POLD1 S869L 42.3 POLD1 R875C 541.36 POLD1 R875C 5.4 POLD1 R875C 0.9 POLD1 R875H 63.1 POLD1 R875C 12.6 POLD1 R875S 72.1 POLD1 R875H 45.9 POLD1 R875C 3.6 POLD1 D877Y 275.7 POLD1 D877N 37.8 POLD1 D877N 455.9 POLD1 D877G 13.5 POLD1 V882L 56.8 POLD1 V882I 11.7 POLD1 I883T 53.2 POLD1 T884I 226.1 POLD1 E886K 1.8 POLD1 E886K 150.5 POLD1 E886K 5.4 POLD1 R889H 3.6 POLD1 R889H 0.9 POLD1 R889H 3.6 POLD1 R889H 6.3 POLD1 R889C 14.4 POLD1 A890V 2.7 POLD1 A890V 6.3 POLD1 A890V 18 POLD1 A890T 73 POLD1 A890V 14.4 POLD1 A890T 753.2 POLD1 A890V 0.9 POLD1 A890V 0 POLD1 D893N 82.9 POLD1 D893N 6.3 POLD1 A895S 4.5 POLD1 G896S 23.4 POLD1 G896S 18.9 POLD1 G896S 9 POLD1 G896D 74.8 POLD1 G896D 237.8 POLD1 G896S 7.2 POLD1 A899T 6.3 POLD1 A899S 13.5 POLD1 A899V 124.3 POLD1 V901M 14.4 POLD1 V901M 13.5 POLD1 E902Q 5.4 POLD1 A904S 16.2 POLD1 E905K 0.9 POLD1 E905K 8.1 POLD1 M907I 23.4 POLD1 M907I 21.6 POLD1 R908K 7.2 POLD1 R908K 64.9 POLD1 R908K 43.2 POLD1 R910Q 3.6 POLD1 R910W 156.8 POLD1 R910Q 18.9 POLD1 R910Q 29.7 POLD1 R910Q 10.8 POLD1 P912S 521.6 POLD1 G913R 2.7 POLD1 G913R 4.5 POLD1 G913R 1.8 POLD1 A915V 91.9 POLD1 D920Y 14.4 POLD1 D920N 0 POLD1 D920N 4.5 POLD1 D920N 5.4 POLD1 D920N 9.9 POLD1 D920N 3.6 POLD1 D920Y 6.3 POLD1 R921H 6.3 POLD1 R921H 45 POLD1 R921C 9.9 POLD1 V922I 17.1 POLD1 P923L 174.8 POLD1 P923S 64.9 POLD1 P923R 15.3 POLD1 P923S 135.1 POLD1 V925M 43.2 POLD1 V925M 2.7 POLD1 V925M 0.9 POLD1 V925G 20.7 POLD1 I926L 1.8 POLD1 I927L 9 POLD1 I927L 5.4 POLD1 S928T 6.3 POLD1 S928T 6.3 POLD1 A930V 386.5 POLD1 G932R 2.7 POLD1 G932V 93.7 POLD1 G932D 130.6 POLD1 G932R 9 POLD1 G932A 10.8 POLD1 V933L 3.6 POLD1 A935T 2.7 POLD1 A935T 6.3 POLD1 A935T 23.4 POLD1 A935V 5.4 POLD1 A935T 5.4 POLD1 A935T 4.5 POLD1 A935T 20.7 POLD1 A935T 0.9 POLD1 M937L 0.9 POLD1 S939A 36 POLD1 S939P 128.8 POLD1 E940K 67.6 POLD1 E940V 5.4 POLD1 D941H 22.5 POLD1 D941Y 15.3 POLD1 D941N 4.5 POLD1 D941G 6.3 POLD1 P942S 2.7 POLD1 P942R 4.5 POLD1 P942L 57.7 POLD1 F944L 56.8 POLD1 V945L 18 POLD1 V945M 2.7 POLD1 V945M 85.6 POLD1 V945M 60.4 POLD1 I952T 3.6 POLD1 I952T 18 POLD1 T954M 3.6 POLD1 T954M 6.3 POLD1 T954M 0.9 POLD1 T954M 155 POLD1 T954M 26.1 POLD1 T954M 4.5 POLD1 Y956H 29.7 POLD1 Q961R 5.4 POLD1 A963S 2.7 POLD1 K964N 10.8 POLD1 K964N 3.6 POLD1 P965F 101.8 POLD1 P965L 112.6 POLD1 L966F 14.4 POLD1 L966V 10.8 POLD1 L967V 0.9 POLD1 L967P 49.5 POLD1 L967P 1.8 POLD1 R968C 3.6 POLD1 R968H 29.7 POLD1 R968C 21.6 POLD1 R968H 68.5 POLD1 R968H 37.8 POLD1 I969M 6.3 POLD1 E971K 237.8 POLD1 E971K 5.4 POLD1 P972S 5.4 POLD1 G975C 60.4 POLD1 E976K 0 POLD1 E976* 9.9 POLD1 E976K 0.9 POLD1 E976D 20.7 POLD1 G977C 70.3 POLD1 R978H 8.1 POLD1 R978C 94.6 POLD1 R978H 3.6 POLD1 A979V 32.4 POLD1 E980V 23.4 POLD1 E980* 163.1 POLD1 E980K 0.9 POLD1 E980K 0.9 POLD1 E980K 9.9 POLD1 A981V 2.7 POLD1 A981S 20.7 POLD1 R985L 25.2 POLD1 R985Q 0.9 POLD1 R985W 0 POLD1 R985Q 3.6 POLD1 G986R 11.7 POLD1 G986R 33.3 POLD1 D987N 52.3 POLD1 D987E 9 POLD1 D987N 7.2 POLD1 H988Y 116.2 POLD1 T989M 4.5 POLD1 T989M 3.6 POLD1 T989M 56.8 POLD1 T989M 6.3 POLD1 R990C 6.3 POLD1 R990H 6.3 POLD1 C991W 6.3 POLD1 C991W 2.7 POLD1 C991Y 10.8 POLD1 T993M 5.4 POLD1 T993M 51.4 POLD1 T993M 87.4 POLD1 L995I 493.7 POLD1 L995F 9 POLD1 T996M 41.4 POLD1 G997S 0.9 POLD1 G997S 5.4 POLD1 G997S 19.8 POLD1 G997S 8.1 POLD1 V999M 18.9 POLD1 G1000S 230.6 POLD1 G1000D 89.2 POLD1 G1001S 2.7 POLD1 G1001S 23.4 POLD1 G1001F 10.8 POLD1 A1004V 65.8 POLD1 A1004T 170.3 POLD1 A1004P 5.4 POLD1 A1004V 14.4 POLD1 A1006I 159.5 POLD1 R1008C 5.4 POLD1 R1008C 2.7 POLD1 R1008H 21.6 POLD1 R1008C 68.5 POLD1 R1009C 493.7 POLD1 R1009L 11.7 POLD1 R1009C 7.2 POLD1 R1009H 4.5 POLD1 R1009C 4.5 POLD1 R1009H 25.2 POLD1 R1009H 3.6 POLD1 R1009L 16.2 POLD1 R1009C 0.9 POLD1 R1009C 4.5 POLD1 N1010T 31.5 POLD1 C1011Y 3.6 POLD1 C1012G 100.9 POLD1 C1012Y 24.3 POLD1 I1013T 4.5 POLD1 I1013T 14.4 POLD1 I1013T 1.8 POLD1 R1016C 31.5 POLD1 R1016H 39.6 POLD1 R1016H 29.7 POLD1 R1016C 6.3 POLD1 R1016H 315.3 POLD1 R1016H 69.4 POLD1 R1016H 636.9 POLD1 R1016H 49.5 POLD1 R1016P 13.5 POLD1 T1017S 3.6 POLD1 S1020N 4.5 POLD1 S1020N 4.5 POLD1 H1021P 1.8 POLD1 Q1022* 10.8 POLD1 G1023R 3.6 POLD1 G1023R 1.8 POLD1 V1025L 18.9 POLD1 V1025L 0 POLD1 C1026S 5.4 POLD1 E1027K 91 POLD1 E1027K 2.7 POLD1 E1027Q 3.6 POLD1 E1027K 8.1 POLD1 F1028L 233.3 POLD1 C1029Y 38.7 POLD1 C1029S 16.2 POLD1 Q1030* 24.3 POLD1 P1031L 313.5 POLD1 R1032Q 0.9 POLD1 R1032Q 0 POLD1 E1033* 11.7 POLD1 E1035Q 48.6 POLD1 E1035Q 9 POLD1 E1035Q 2.7 POLD1 E1035Q 9 POLD1 Y1037C 34.2 POLD1 Q1038E 30.6 POLD1 Q1038E 18.9 POLD1 E1040A 472.56 POLD1 E1040Q 14.4 POLD1 E1040K 77.5 POLD1 V1041A 16.2 POLD1 V1041L 5.4 POLD1 L1044V 48.6 POLD1 A1046V 450.5 POLD1 A1046G 4.5 POLD1 A1046V 30.6 POLD1 E1048K 38.7 POLD1 E1049K 132.4 POLD1 E1049K 1.8 POLD1 E1049K 52.3 POLD1 R1050C 541.36 POLD1 R1050C 500 POLD1 R1050S 29.7 POLD1 R1050H 5.4 POLD1 S1052* 32.4 POLD1 S1052P 5.4 POLD1 S1052W 178.4 POLD1 S1052L 3.6 POLD1 S1052L 2.7 POLD1 S1052L 20.7 POLD1 S1052L 9 POLD1 R1053C 541.36 POLD1 R1053H 3.6 POLD1 R1053H 2.7 POLD1 R1053L 8.1 POLD1 W1055* 10.8 POLD1 W1055V 23.4 POLD1 T1056S 35.1 POLD1 T1056K 1.8 POLD1 Q1057R 6.3 POLD1 C1058Y 13.5 POLD1 Q1059* 1.8 POLD1 R1060C 4.5 POLD1 R1060H 33.3 POLD1 R1060H 19.8 POLD1 R1060H 32.4 POLD1 G1063C 15.3 POLD1 G1063C 69.4 POLD1 H1066R 40.5 POLD1 H1066D 73.9 POLD1 H1066Y 31.5 POLD1 H1066R 4.5 POLD1 E1067D 9.9 POLD1 E1067V 36 POLD1 D1068N 14.4 POLD1 I1070M 22.5 POLD1 S1073T 3.6 POLD1 R1074Q 19.8 POLD1 R1074W 18.9 POLD1 R1074W 213.5 POLD1 R1074Q 9.9 POLD1 R1074W 21.6 POLD1 D1075H 76.6 POLD1 P1077S 34.2 POLD1 P1077A 5.4 POLD1 P1077A 11.7 POLD1 P1077L 4.5 POLD1 F1079L 0.9 POLD1 F1079L 38.7 POLD1 F1079L 29.7 POLD1 F1079L 9.9 POLD1 Y1080H 11.7 POLD1 M1081K 0 POLD1 M1081I 1.8 POLD1 R1082H 2.7 POLD1 R1082C 11.7 POLD1 R1082H 43.2 POLD1 K1083N 16.2 POLD1 V1085L 0.9 POLD1 R1086W 238.18 POLD1 R1086W 472.56 POLD1 R1086W 7.2 POLD1 R1086W 13.5 POLD1 R1086W 12.6 POLD1 R1086W 5.4 POLD1 R1086W 4.5 POLD1 R1086P 7.2 POLD1 E1090K 2.7 POLD1 E1090K 3.6 POLD1 D1091N 0.9 POLD1 D1091N 18.9 POLD1 D1091N 7.2 POLD1 E1093K 381.1 POLD1 E1093K 2.7 POLD1 E1093K 402.7 POLD1 E1093K 3.6 POLD1 Q1094H 13.5 POLD1 R1097G 119.8 POLD1 R1097Q 5.4 POLD1 R1097Q 5.4 POLD1 R1097W 5.4 POLD1 R1097W 2.7 POLD1 R1097W 70.3 POLD1 R1097Q 0 POLD1 R1098C 4.5 POLD1 R1098C 18.9 POLD1 R1098C 70.3 POLD1 R1098L 3.6 POLD1 R1098C 6.3 POLD1 F1099L 7.2 POLD1 F1099L 5.4 POLD1 G1100V 7.2 POLD1 P1101S 3.6 POLD1 P1101S 79.3 POLD1 P1101L 20.7 POLD1 P1101L 71.2 POLD1 P1102L 0.9 POLD1 G1103* 4.5 POLD1 G1103A 12.6 POLD1 P1104T 4.5 POLD1 P1104L 164.9 POLD1 E1105* 5.4 POLD1 A1106D 1.8 POLD1 W1107C 95.5 POLD1 D987fs*58 78.4 POLD1 D819fs*69 45.9 POLD1 R180fs*72 40.5 POLD1 M727del 32.4 POLD1 M457fs*179 1.8 POLD1 D987fs*58 45 POLD1 G724fs*15 13.5 POLD1 splice site 590 − 2A > G 0 POLD1 Y147fs*22 9 POLD1 L543fs*92 28.8 POLD1 D987fs*58 91 POLD1 splice site 316 + 1delG 8.1 POLD1 R823fs*68 6.3 POLD1 P1102fs*7+ 36.9 POLD1 E346fs*47 54.1 POLD1 P116fs*53 51.4 POLD1 D987fs*58 214.4 POLD1 L188fs*89 44.1 POLD1 I172fs*80 2.7 POLD1 D987fs*41 16.2 POLD1 E795fs*1 3.6 POLD1 T642fs*6 107.2 POLD1 P116fs*53 47.7 POLD1 A28_P29 > DAP 3.6 POLD1 F34fs*9 24.3 POLD1 splice site 2954 − 2insT 26.1 POLD1 P116fs*53 30.6 POLD1 D987fs*58 75.7 POLD1 D987fs*58 15.3 POLD1 R358fs*27 7.2 POLD1 E159fs*10 13.5 POLD1 C338del 38.7 POLD1 splice site 2251 − 1G > A 7.2 POLD1 D987fs*58 37.8 POLD1 splice site 1495 − 1G > A 3.6 POLD1 V111fs*58 42.3 POLD1 R823_M824insPDAHDR 12.6 POLD1 C1029fs*1 2.7 POLD1 W265fs*1 12.6 POLD1 splice site 1893 − 2A > G 278.4 POLD1 P116fs*53 29.7 POLD1 F209fs*67 63.1 POLD1 D987fs*58 39.6 POLD1 R180fs*3 56.8 POLD1 P404fs*74 3.6 POLD1 splice site 1384 − 2A > G 43.2 POLD1 P116fs*53 56.8 POLD1 V61fs*42 5.4 POLD1 L839fs*20 28.8 POLD1 P116fs*53 85.6 POLD1 D987fs*58 42.3 POLD1 D987fs*58 31.5 POLD1 splice site 1686 + 2T > C 60.4 POLD1 D987fs*41 39.6 POLD1 P116fs*53 37.8 POLD1 K486del 0 POLD1 splice site 1243 − 1G > T 21.6 POLD1 D987fs*58 68.5 POLD1 D987fs*58 57.7 POLD1 D515fs*16 9 POLD1 splice site 1137 + 1G > A 5.4 POLD1 splice site 203 − 1G > A 5.4 POLD1 Y801fs*1 9.9 POLD1 R1032fs*13 58.6 POLD1 P1102fs*7+ 7.2 POLD1 D987fs*58 40.5 POLD1 P1102fs*7+ 1.8 POLD1 E279del 6.3 POLD1 R180fs*72 17.1 POLD1 D987fs*58 57.7 POLD1 S719fs*19 7.2 POLD1 splice site 1137 + 2T > C 5.4 POLD1 D987fs*58 36 POLD1 P942fs*100 6.3 POLD1 E279del 0 POLD1 E196_S197del 99.1 POLD1 E279del 9 POLD1 R817fs*67 5.4 POLD1 P116fs*53 66.7 POLD1 R253fs*23 5.4 POLD1 T642fs*6 7.2 POLD1 D987fs*58 5.4 POLD1 splice site 1494 + 2T > A 7.2 POLD1 D987fs*58 70.3 POLD1 K931_Y936del 5.4 POLD1 D987fs*58 65.8 POLD1 splice site 1495 − 1G > A 14.4 POLD1 P116fs*53 109 POLD1 D987fs*58 57.7 POLD1 P116fs*53 36 POLD1 D987fs*58 45.9 POLD1 D987fs*58 48.6 POLD1 D987fs*58 122.5 POLD1 P116fs*53 44.1 POLD1 V111fs*58 63.1 POLD1 E279del 8.1 POLD1 E346fs*47 59.5 POLD1 T183fs*93 4.5 POLD1 splice site 2953 + 1G > A 27.9 POLD1 splice site 3116_3120 + 11delAGGAGGTGAGAGGGCC 13.5 POLD1 P1102fs*7+ 49.5 POLD1 P116fs*17 53.2 POLD1 D987fs*41 26.1 POLD1 P116fs*53 40.5 POLD1 T642fs*97 73 POLD1 D987fs*58 27 POLD1 splice site 2155 − 1G > T 23.4 POLD1 D987fs*58 43.2 POLD1 K897fs*54 0.9 POLD1 P154fs*15 96.4 POLD1 Q628fs*5 96.4 POLD1 P1102fs*7+ 14.4 POLD1 D987fs*58 69.4 POLD1 splice site 1893 − 2A > T 28.8 POLD1 splice site 1892 + 1G > A 38.7 POLD1 R180fs*3 11.7 POLD1 splice site 317 − 9_333del26 5.4 POLD1 K732fs*156 1.8 POLD1 I212fs*35 2.7 POLD1 D987fs*58 75.7 POLD1 E42_E45del 9.9 POLD1 splice site 1495 − 1G > A 13.5 POLD1 D987fs*58 153.2 POLD1 A979fs*66 84.7 POLD1 R180fs*72 43.2 POLD1 D987fs*58 70.3 POLD1 S816del 9 POLD1 P116fs*53 67.6 POLD1 E279del 9.9 POLD1 R180fs*3 55 POLD1 D987fs*58 70.3 POLD1 splice site 2953 + 1G > T 4.5 POLD1 R180fs*3 279.3 POLD1 splice site 2389 − 1G > A 5.4 POLD1 D987fs*58 17.1 POLD1 S605del 14.4 POLD1 splice site 971 − 1G > T 73 POLD1 D987fs*41 42.3 POLD1 D987fs*58 64 POLD1 P116fs*53 80.2 POLD1 W1055fs*50 68.5 POLD1 D987fs*58 36.9 POLD1 D987fs*58 32.4 POLD1 D987fs*58 35.1 POLD1 D987fs*58 50.5 POLD1 splice site 2953 + 1G > A 21.6 POLD1 D987fs*58 14.4 POLD1 splice site 758 + 1G > T 26.1 POLD1 D987fs*58 42.3 POLD1 V111fs*58 9.9 POLD1 S816del 31.5 POLD1 E742fs*8 2.7 POLD1 F814_S815 > S 4.5 POLD1 R180fs*3 67.6 POLD1 P1102fs*7+ 12.6 POLD1 D76fs*25 17.1 POLD1 D987fs*58 118.9 POLD1 D987fs*58 40.5 POLD1 D987fs*58 8.1 POLD1 R180fs*3 45 POLD1 A18_G20del 2.7 POLD1 splice site 759 − 1G > T 36.9 POLD1 R180fs*72 109 POLD1 S816del 11.7 POLD1 L966fs*75 6.3 POLD1 R667fs*28 4.5 POLD1 D987fs*58 49.5 POLD1 P116fs*53 55.9 POLD1 K648fs*46 17.1 POLD1 splice site 1495 − 1G > T 20.7 POLD1 P115fs*19 36 POLD1 D987fs*41 61.3 POLD1 D987fs*58 36 POLD1 L681fs*13 22.5 POLD1 V933fs*21 70.3 POLD1 E346fs*47 87.4 POLD1 L839fs*49 48.6 POLD1 splice site 2564 + 1G > A 4.5 POLD1 splice site 316 + 1G > T 50.5 POLD1 S605del 8.1 POLD1 E346fs*47 42.3 POLD1 S605del 80.2 POLD1 S816del 6.3 POLD1 P116fs*53 78.4 POLD1 Q707fs*21 3.6 POLD1 D987fs*58 164.9 POLD1 P116fs*53 16.2 POLD1 D987fs*58 70.3 POLD1 D987fs*58 159.5 POLD1 splice site 2821 − 1G > A 7.2 POLD1 S847_R850 > C 2.7 POLD1 splice site 841 − 2A > G 16.2 POLD1 R823fs*65 16.2 POLD1 splice site 2821 − 1G > T 25.2 POLD1 S816del 23.4 POLD1 P116fs*53 76.6 POLD1 splice site 1495 − 1G > A 7.2 POLD1 T91fs*78 2.7 POLD1 D987fs*58 68.5 POLD1 S240_P243del 19.8 POLD1 D987fs*58 40.5 POLD1 D987fs*58 75.7 POLD1 D987fs*58 12.6 POLD1 D987fs*58 52.3 POLD1 splice site 2821 − 1G > C 27 POLD1 D987fs*58 65.8 POLD1 splice site 2251 − 1G > A 7.2 POLD1 E47fs*44 5.4 POLD1 L1089fs*20+ 62.2 POLD1 splice site 2250 + 1G > A 3.6 POLD1 splice site 3218 + 2T > G 10.8 POLD1 S816del 7.2 POLD1 E57del 3.6 POLD1 S783fs*106 5.4 POLD1 R180fs*3 19.8 POLD1 A28_P29 > VAP 18.9 POLD1 D987fs*58 69.4 POLD1 P116fs*53 45

TABLE 6 POLE and POLD Mutations Specimen Driver Mutation ID Tumour Type Gene Mutation Burden Stage Status Drivers POLE 1 Colorectal Carcinoma POLE Y458H 342 Adult New 2 Brain PNET POLE Y458H 85.02 Pediatric New 3 Colon adenocarcinoma (CRC) POLE Y458C 753.2 Adult New 4 Colon adenocarcinoma (CRC) POLE Y458C 13.5 Adult New 5 Colon adenocarcinoma (CRC) POLE V411L 703.6 Adult Known 6 Brain glioblastoma POLE V411L 699 Pediatric Known 7 Ovary carcinosarcoma POLE V411L 650.5 Adult Known 8 Uterus endometrial POLE V411L 636.9 Adult Known adenocarcinoma (NOS) 9 Brain glioblastoma (GBM) POLE V411L 578.4 Adult Known 10 Kidney urothelial carcinoma POLE V411L 500 Adult Known 11 Prostate neuroendocrine POLE V411L 371.2 Adult Known carcinoma 12 Colon adenocarcinoma (CRC) POLE V411L 360.4 Adult Known 13 Uterus endometrial POLE V411L 345.9 Adult Known adenocarcinoma endometrioid 14 Uterus endometrial POLE V411L 324.3 Adult Known adenocarcinoma (NOS) 15 Brain glioma (NOS) POLE V411L 315.3 Adult Known 16 Ovary epithelial carcinoma (NOS) POLE V411L 309.9 Adult Known 17 Prostate acinar adenocarcinoma POLE V411L 301.8 Adult Known 18 Prostate neuroendocrine POLE V411L 279.3 Adult Known carcinoma 19 Prostate acinar adenocarcinoma POLE V411L 245.9 Adult Known 20 Rectum adenocarcinoma (CRC) POLE V411L 240.5 Adult Known 21 Uterus endometrial POLE V411L 238.7 Adult Known adenocarcinoma (NOS) 22 Uterus endometrial POLE V411L 187.4 Adult Known adenocarcinoma (NOS) 23 Colon adenocarcinoma (CRC) POLE V411L 136 Adult Known 24 Colon adenocarcinoma (CRC) POLE V411L 120.7 Adult Known 25 Colon adenocarcinoma (CRC) POLE V411L 116.2 Adult Known 26 Ovary clear cell carcinoma POLE V411L 105.4 Adult Known 27 Colon adenocarcinoma (CRC) POLE V411L 104.5 Adult Known 28 Rectum adenocarcinoma (CRC) POLE V411L 100 Adult Known 29 Uterus carcinosarcoma POLE V411L 77.5 Adult Known 30 Brain glioblastoma (GBM) POLE V411L 45 Adult Known 31 Colon adenocarcinoma (CRC) POLE V411L 39.6 Adult Known 32 Brain glioblastoma (GBM) POLE S4611 447.8 Adult Known 33 Brain glioblastoma POLE S461P 496.24 Pediatric Known 34 Brain glioblastoma POLE S461P 208 Pediatric Known 35 Unknown primary carcinoma POLE S459F 311.7 Adult Known (NOS) 36 Brain glioblastoma POLE S459F 295 Pediatric Known 37 Rectum adenocarcinoma (CRC) POLE S459F 146.8 Adult Known 38 Brain glioblastoma (GBM) POLE S459F 117.1 Adult Known 39 Uterus endometrial POLE S459F 102.7 Adult Known adenocarcinoma (NOS) 40 Duodenum adenocarcinoma POLE S459F 36.9 Adult Known 41 Brain glioblastoma (GBM) POLE S297F 386.5 Adult Known 42 Brain glioblastoma POLE S297F 218 Pediatric Known 43 Skin melanoma POLE 5297F 115.3 Adult Known 44 Uterus endometrial POLE 5297F 39.6 Adult Known adenocarcinoma (NOS) 45 Colorectal Carcinoma POLE P436S 433 Adult New 46 Brain glioblastoma (GBM) POLE P436S 333.3 Adult New 47 Brain glioblastoma POLE P436S 318.06 Pediatric New 48 Brain glioblastoma POLE P436S 302 Pediatric New 49 Osteochondroma POLE P436S 195 Pediatric New 50 Colon adenocarcinoma (CRC) POLE P436R 493.7 Adult New 51 Brain glioblastoma POLE P436H 541.36 Pediatric New 52 Brain glioblastoma POLE P436H 532 Pediatric New 53 Brain glioblastoma POLE P436H 409.18 Pediatric New 54 Brain glioblastoma POLE P436H 359 Pediatric New 55 Skin basal cell carcinoma POLE P286S 58.6 Adult New 56 Unknown primary melanoma POLE P286S 42.3 Adult New 57 Uterus endometrial POLE P286R 493.7 Adult Known adenocarcinoma (NOS) 58 Colon adenocarcinoma (CRC) POLE P286R 479.3 Adult Known 59 Brain glioblastoma (GBM) POLE P286R 450.5 Adult Known 60 Colon adenocarcinoma (CRC) POLE P286R 394.6 Adult Known 61 Colon adenocarcinoma (CRC) POLE P286R 342.3 Adult Known 62 Colon adenocarcinoma (CRC) POLE P286R 305.4 Adult Known 63 Colon adenocarcinoma (CRC) POLE P286R 259.5 Adult Known 64 Colon adenocarcinoma (CRC) POLE P286R 245 Adult Known 65 Brain glioblastoma (GBM) POLE P286R 227.9 Adult Known 66 Colon adenocarcinoma (CRC) POLE P286R 226.1 Adult Known 67 Rectum adenocarcinoma (CRC) POLE P286R 223.4 Adult Known 68 Colon adenocarcinoma (CRC) POLE P286R 216.2 Adult Known 69 Uterus endometrial POLE P286R 208.1 Adult Known adenocarcinoma (NOS) 70 Uterus endometrial POLE P286R 203.6 Adult Known adenocarcinoma (NOS) 71 Colon adenocarcinoma (CRC) POLE P286R 172.1 Adult Known 72 Uterus carcinosarcoma POLE P286R 168.5 Adult Known 73 Ovary neuroendocrine carcinoma POLE P286R 163.1 Adult Known 74 Rectum adenocarcinoma (CRC) POLE P286R 162.2 Adult Known 75 Colon adenocarcinoma (CRC) POLE P286R 155 Adult Known 76 Uterus endometrial POLE P286R 153.2 Adult Known adenocarcinoma (NOS) 77 Uterus endometrial POLE P286R 145.9 Adult Known adenocarcinoma (NOS) 78 Colon adenocarcinoma (CRC) POLE P286R 133.3 Adult Known 79 Uterus endometrial POLE P286R 123.4 Adult Known adenocarcinoma (NOS) 80 Pancreas ductal adenocarcinoma POLE P286R 100.9 Adult Known 81 Uterus endometrial POLE P286R 84.7 Adult Known adenocarcinoma (NOS) 82 rectum neuroendocrine carcinoma POLE P286R 80.2 Adult Known 83 Colon adenocarcinoma (CRC) POLE P286R 62.2 Adult Known 84 Brain glioblastoma (GBM) POLE P286H 212 Adult New 85 Brain glioblastoma (GBM) POLE M444K 116.2 Adult Known 86 Colon adenocarcinoma (CRC) POLE M444K 91.9 Adult Known 87 Uterus neuroendocrine carcinoma POLE M444K 32.4 Adult Known 88 Colorectal carcinoma POLE L424P 590 Pediatric New 89 Brain glioblastoma POLE L424I 336 Pediatric Known 90 Brain glioblastoma (GBM) POLE L424I 160.4 Adult Known 91 Lung adenocarcinoma POLE L424I 154.1 Adult Known 92 Liver cholangiocarcinoma POLE L424I 47.7 Adult Known 93 Brain glioblastoma POLE F367L 472.56 Pediatric New 94 Brain glioblastoma POLE F367L 238.18 Pediatric New 95 Unknown primary squamous POLE E978K 80.2 Adult New cell carcinoma (SCC) 96 Brain glioblastoma POLE E978G 608.02 Pediatric New 97 Uterus carcinosarcoma POLE E978G 26.1 Adult New 98 Appendix adenocarcinoma POLE D368N 36 Adult New 99 Lung adenocarcinoma POLE D275V 43.2 Adult Known 100 Brain glioblastoma (GBM) POLE D275V 17.1 Adult Known 101 Pancreas ductal adenocarcinoma POLE D275G 318.9 Adult Known 102 Brain glioblastoma (GBM) POLE D275A 226.1 Adult Known 103 Rectum adenocarcinoma (CRC) POLE C810G 854.1 Adult New 104 Brain glioblastoma (GBM) POLE C810G 154.1 Adult New 105 Lung squamous cell carcinoma POLE A463V 231.5 Adult New (SCC) 106 Uterus endometrial POLE A463V 55.9 Adult New adenocarcinoma (NOS) 107 Brain oligodendroglioma POLE A463T 809 Adult New 108 Brain glioblastoma POLE A463D 532 Pediatric New 109 Brain glioblastoma POLE A463D 359 Pediatric New 110 Colon adenocarcinoma (CRC) POLE A456P 373.9 Adult Known 111 Colon adenocarcinoma (CRC) POLE A456P 314.4 Adult Known 112 Colon adenocarcinoma (CRC) POLE A456P 233.3 Adult Known 113 Uterus endometrial POLE A456P 226.1 Adult Known adenocarcinoma (NOS) 114 Colon adenocarcinoma (CRC) POLE A456P 200 Adult Known 115 Ovary endometrioid POLE A456P 69.4 Adult Known adenocarcinoma 116 Cervix squamous cell carcinoma POLE A456P 33.3 Adult Known (SCC) 117 Brain glioblastoma (GBM) POLE A288V 578.4 Adult New 118 Brain glioblastoma (GBM) POLE A288V 386.5 Adult New 119 Stomach neuroendocrine POLE A288V 155.9 Adult New carcinoma Drivers POLD1 1 Colon adenocarcinoma (CRC) POLD1 R689W 259.5 Adult Known 2 Brain oligodendroglioma POLD1 R689W 585.6 Adult Known 3 Brain glioblastoma (GBM) POLD1 R689W 578.4 Adult Known 4 Brain glioblastoma (GBM) POLD1 R689W 447.8 Adult Known 5 Colon adenocarcinoma (CRC) POLD1 R689W 12.6 Adult Known 6 Vulva squamous cell carcinoma POLD1 R1016H 39.6 Adult New (SCC) 7 Colon adenocarcinoma (CRC) POLD1 R1016H 29.7 Adult New 8 Brain glioma (NOS) POLD1 R1016H 315.3 Adult New 9 Unknown primary POLD1 R1016H 69.4 Adult New adenocarcinoma 10 Uterus endometrial POLD1 R1016H 636.9 Adult New adenocarcinoma (NOS) 11 Colon adenocarcinoma (CRC) POLD1 R1016H 49.5 Adult New 12 Brain medullobastoma POLD1 L632M 141 Pediatric New 13 Brain glioblastoma POLD1 L632M 228 Pediatric New 14 Brain glioblastoma POLD1 L632M 296 Pediatric New 15 Brain Glioblastoma POLD1 L632M 121 Pediatric New 16 Brain PNET POLD1 L606M 196 Pediatric Known 17 Brain PNET POLD1 L606M 295 Pediatric Known 18 Brain glioblastoma (GBM) POLD1 L606M 228.8 Adult Known 19 Brain glioblastoma (GBM) POLD1 L606M 51.4 Adult Known 20 Uterus carcinosarcoma POLD1 L606M 311.7 Adult Known 21 Brain PNET POLD1 E318K 182 Pediatric New 22 Lung non-small cell lung POLD1 E318K 13.5 Adult New carcinoma (NOS) 23 Colon adenocarcinoma (CRC) POLD1 E245K 76.6 Adult New 24 Liver cholangiocarcinoma POLD1 E245K 122.5 Adult New 25 Colon adenocarcinoma (CRC) POLD1 E245K 85.6 Adult New

TABLE 7 Tumour samples with germline replication repair deficiency analysed by exome sequencing Somatic Somatic Sample Tissue Germline POLE POLD1 TMB 1 Brain bMMRD X UHM 2 Brain bMMRD X UHM 3 Brain bMMRD X UHM 4 Brain bMMRD X UHM 5 Brain bMMRD X UHM 6 Colon bMMRD HM 7 Colon bMMRD HM 8 Colon bMMRD HM 9 Colon bMMRD HM 10 Colon bMMRD HM 11 Brain bMMRD UHM 12 Leukemia bMMRD HM 13 Brain bMMRD X UHM 14 Brain bMMRD X UHM 15 Brain bMMRD X UHM 16 Brain bMMRD X HM 17 Brain bMMRD X UHM 18 Brain bMMRD X UHM 19 Brain bMMRD X UHM 20 Brain bMMRD X UHM 21 Small Intestine bMMRD HM 22 Brain POLE UHM 23 Brain bMMRD X UHM 24 Brain Lynch HM 25 Breast bMMRD HM 26 Colon bMMRD X UHM 27 Colon bMMRD X UHM 28 Brain bMMRD X UHM 29 Brain bMMRD X UHM 30 Brain bMMRD X UHM 31 Leukemia bMMRD HM 32 Brain bMMRD X UHM 33 Brain bMMRD X UHM 34 Brain bMMRD X UHM 35 Brain bMMRD X UHM 36 Brain bMMRD X UHM 37 Brain bMMRD X UHM 38 Brain bMMRD X UHM 39 Brain bMMRD X UHM 40 Leukemia bMMRD HM 41 Brain bMMRD X UHM 42 Brain bMMRD X UHM 43 Brain bMMRD X UHM 44 Brain bMMRD X UHM 45 Colon POLE UHM 46 Brain Lynch UHM 47 Colon bMMRD X UHM 48 Colon bMMRD X UHM 49 Adrenal Gland bMMRD HM 50 Bone bMMRD X UHM 51 Brain bMMRD X UHM 52 Brain bMMRD X UHM 53 Brain bMMRD X UHM 54 Brain bMMRD X UHM 55 Brain bMMRD X UHM 56 Brain bMMRD X HM 

What is claimed is:
 1. A method of profiling a tumour, the method comprising: sequencing nucleic acid from a sample obtained from the tumour to obtain nucleic acid sequences; identifying mutations in the nucleic acid sequences relative to a reference sequence obtained from a healthy tissue, wherein each of said mutations is defined with respect to the pyrimidine of a base pair; determining for the tumour, using the mutations, relative proportions of each of 96 mutation types, wherein the 96 mutation types are defined as each of six possible pyrimidine base changes C>A, C>G, C>T, T>A, T>C, or T>G in the context of each of four possible nucleotides (A, C, G, or T) at the position immediately 5′ to the mutation and each of four possible nucleotides (A, C, G, or T) at the position immediately 3′ to the mutation, comparing the relative proportions of at least 48 of the 96 mutation types for the tumour to corresponding mean proportions for the mutation types for clusters 1 to 3 depicted in Table 1A: TABLE 1A Mean Proportion for Clusters Mut 5′ 3′ 1 2 3 C > A A A 6.7E-05 0.001303 0.00119 C > A A C 0.002389 0.003823 0.003755 C > A A G 0 0.00119 0.000792 C > A A T 0.009421 0.002499 0.006822 C > A C A 0.003067 0.00708 0.003819 C > A C C 0.004592 0.011953 0.002766 C > A C G 0.00161 0.007254 0.002327 C > A C T 0.037945 0.029925 0.014682 C > A G A 0.001125 0.002937 0.002638 C > A G C 0.005352 0.004551 0.002743 C > A G G 0.001282 0.002862 0.00103 C > A G T 0.032471 0.008021 0.008659 C > A T A 0.002468 0.002416 0.030984 C > A T C 0.006074 0.004215 0.008444 C > A T G 0.000658 0.001553 0.003072 C > A T T 0.041834 0.006319 0.190779 C > G A A 0.000569 0.001268 0.000741 C > G A C 0.000114 0.002475 0.000842 C > G A G 0.000251 0.001332 0.000468 C > G A T 0 0.001943 0.000872 C > G C A 0.00032 0.00168 0.00023 C > G C C 0.000559 0.002767 0.001219 C > G C G 0.000379 0.003067 0.000998 C > G C T 0.000202 0.001803 0.001843 C > G G A 0.000238 0.001606 0.000929 C > G G C 0.000404 0.003352 0.001803 C > G G G 5.71E-05 0.001414 0.00021 C > G G T 7.05E-05 0.001991 0.001271 C > G T A 0 0.001682 0.000244 C > G T C 0.000343 0.002414 0.000753 C > G T G 0 0.001217 0.000503 C > G T T 0.001043 0.004031 0.000942 C > T A A 0.009098 0.015807 0.002356 C > T A C 0.021673 0.014512 0.007633 C > T A G 0.080822 0.098997 0.029695 C > T A T 0.01273 0.008974 0.006131 C > T C A 0.005011 0.009953 0.002726 C > T C C 0.013316 0.012312 0.003786 C > T C G 0.075944 0.103416 0.024621 C > T C T 0.013479 0.012866 0.006972 C > T G A 0.024254 0.028917 0.005288 C > T G C 0.059279 0.046541 0.024879 C > T G G 0.222447 0.148718 0.070994 C > T G T 0.038951 0.030229 0.021253 C > T T A 0.007213 0.010064 0.004939 C > T T C 0.028449 0.011086 0.016469 C > T T G 0.078732 0.052801 0.219143 C > T T T 0.027756 0.008177 0.02943 T > A A A 0.000295 0.001176 0.000209 T > A A C 0.001226 0.003815 0.000852 T > A A G 0.0004 0.001401 0.000458 T > A A T 0.001352 0.002917 0.001796 T > A C A 0 0.000781 0.000352 T > A C C 0.000369 0.002485 0.001001 T > A C G 0.0005 0.004052 0.001149 T > A C T 0.000809 0.001423 0.000821 T > A G A 0 0.0012 0.000226 T > A G C 0.002031 0.002942 0.000182 T > A G G 0.00019 0.003729 0.000445 T > A G T 0.000321 0.00107 0.000202 T > A T A 0 0.000781 0.000138 T > A T C 0.001246 0.00113 0.000725 T > A T G 0.000229 0.000808 0.00015 T > A T T 0.000267 0.000915 0.00279 T > C A A 0.006228 0.010066 0.004417 T > C A C 0.005627 0.00713 0.004952 T > C A G 0.004509 0.021637 0.005165 T > C A T 0.00324 0.007429 0.004211 T > C C A 0.001378 0.007187 0.000923 T > C C C 0.004677 0.009799 0.003691 T > C C G 0.003798 0.025847 0.003957 T > C C T 0.002998 0.009297 0.003321 T > C G A 0.014476 0.017372 0.01155 T > C G C 0.012038 0.011524 0.015514 T > C G G 0.012999 0.020376 0.008717 T > C G T 0.008026 0.011538 0.012768 T > C T A 0.002157 0.00638 0.002805 T > C T C 0.003458 0.007139 0.00666 T > C T G 0.002926 0.009561 0.005562 T > C T T 0.002948 0.005824 0.005819 T > G A A 6.7E-05 0.000486 0.001478 T > G A C 0.001077 0.001094 0.003775 T > G A G 0.000499 0.001114 0.001846 T > G A T 0.003055 0.00097 0.011338 T > G C A 0.000196 0.00167 0.002233 T > G C C 0.000731 0.004141 0.001955 T > G C G 0.000628 0.00923 0.003836 T > G C T 0.003131 0.005064 0.015868 T > G G A 0.000365 0.000912 0.001333 T > G G C 0.001875 0.001146 0.002466 T > G G G 0.001543 0.002292 0.00168 T > G G T 0.003994 0.003071 0.004097 T > G T A 0 0.000364 0.004756 T > G T C 0.001569 0.001778 0.006492 T > G T G 0.000386 0.001434 0.001971 T > G T T 0.006112 0.001191 0.048665

assigning the tumour to one of the clusters 1 to 3 based on the comparing, wherein: cluster 1 is indicative of an ultra-hypermutant tumour with microsatellite stability, a germline MMR gene mutation, or a POLE gene mutation secondary an MMR mutation; cluster 2 is indicative of a hypermutant tumour with microsatellite instability, or an early MMR gene mutation; and cluster 3 is indicative of an ultra-hypermutant tumour with microsatellite stability, an early POLE gene mutation, or an MMR gene mutation secondary to a POLE gene mutation; and after assigning the tumor to one of the clusters 1 to 3, administering to the patient an immunotherapy or an immune checkpoint inhibitor.
 2. The method of claim 1, wherein the comparing is carried out using the relative proportions of the 96 mutation types determined for the tumour.
 3. The method of claim 1, wherein the step of assigning is carried out by matching the relative proportions determined for the tumour to one of the clusters by highest cosine similarity.
 4. The method of claim 3, wherein the matching requires a minimum cosine similarity of 0.75.
 5. The method of claim 1, wherein the at least one tumour characteristic is further determined based on the presence of one or more driver mutations as defined in Table 6: TABLE 6 Specimen Driver Mutation ID Tumour Type Gene Mutation Burden Stage Status Drivers POLE 1 Colorectal Carcinoma POLE Y458H 342 Adult New 2 Brain PNET POLE Y458H 85.02 Pediatric New 3 Colon adenocarcinoma (CRC) POLE Y458C 753.2 Adult New 4 Colon adenocarcinoma (CRC) POLE Y458C 13.5 Adult New 5 Colon adenocarcinoma (CRC) POLE V411L 703.6 Adult Known 6 Brain glioblastoma POLE V411L 699 Pediatric Known 7 Ovary carcinosarcoma POLE V411L 650.5 Adult Known 8 Uterus endometrial POLE V411L 636.9 Adult Known adenocarcinoma (NOS) 9 Brain glioblastoma (GBM) POLE V411L 578.4 Adult Known 10 Kidney urothelial carcinoma POLE V411L 500 Adult Known 11 Prostate neuroendocrine POLE V411L 371.2 Adult Known carcinoma 12 Colon adenocarcinoma (CRC) POLE V411L 360.4 Adult Known 13 Uterus endometrial POLE V411L 345.9 Adult Known adenocarcinoma endometrioid 14 Uterus endometrial POLE V411L 324.3 Adult Known adenocarcinoma (NOS) 15 Brain glioma (NOS) POLE V411L 315.3 Adult Known 16 Ovary epithelial carcinoma POLE V411L 309.9 Adult Known (NOS) 17 Prostate acinar POLE V411L 301.8 Adult Known adenocarcinoma 18 Prostate neuroendocrine POLE V411L 279.3 Adult Known carcinoma 19 Prostate acinar POLE V411L 245.9 Adult Known adenocarcinoma 20 Rectum adenocarcinoma POLE V411L 240.5 Adult Known (CRC) 21 Uterus endometrial POLE V411L 238.7 Adult Known adenocarcinoma (NOS) 22 Uterus endometrial POLE V411L 187.4 Adult Known adenocarcinoma (NOS) 23 Colon adenocarcinoma (CRC) POLE V411L 136 Adult Known 24 Colon adenocarcinoma (CRC) POLE V411L 120.7 Adult Known 25 Colon adenocarcinoma (CRC) POLE V411L 116.2 Adult Known 26 Ovary clear cell carcinoma POLE V411L 105.4 Adult Known 27 Colon adenocarcinoma (CRC) POLE V411L 104.5 Adult Known 28 Rectum adenocarcinoma POLE V411L 100 Adult Known (CRC) 29 Uterus carcinosarcoma POLE V411L 77.5 Adult Known 30 Brain glioblastoma (GBM) POLE V411L 45 Adult Known 31 Colon adenocarcinoma (CRC) POLE V411L 39.6 Adult Known 32 Brain glioblastoma (GBM) POLE S461T 447.8 Adult Known 33 Brain glioblastoma POLE S461P 496.24 Pediatric Known 34 Brain glioblastoma POLE S461P 208 Pediatric Known 35 Unknown primary carcinoma (NOS) POLE S459F 311.7 Adult Known 36 Brain glioblastoma POLE S459F 295 Pediatric Known 37 Rectum adenocarcinoma (CRC) POLE S459F 146.8 Adult Known 38 Brain glioblastoma (GBM) POLE S459F 117.1 Adult Known 39 Uterus endometrial POLE S459F 102.7 Adult Known adenocarcinoma (NOS) 40 Duodenum adenocarcinoma POLE S459F 36.9 Adult Known 41 Brain glioblastoma (GBM) POLE S297F 386.5 Adult Known 42 Brain glioblastoma POLE S297F 218 Pediatric Known 43 Skin melanoma POLE S297F 115.3 Adult Known 44 Uterus endometrial POLE S297F 39.6 Adult Known adenocarcinoma (NOS) 45 Colorectal Carcinoma POLE P436S 433 Adult New 46 Brain glioblastoma (GBM) POLE P436S 333.3 Adult New 47 Brain glioblastoma POLE P436S 318.06 Pediatric New 48 Brain glioblastoma POLE P436S 302 Pediatric New 49 Osteochondroma POLE P436S 195 Pediatric New 50 Colon adenocarcinoma (CRC) POLE P436R 493.7 Adult New 51 Brain glioblastoma POLE P436H 541.36 Pediatric New 52 Brain glioblastoma POLE P436H 532 Pediatric New 53 Brain glioblastoma POLE P436H 409.18 Pediatric New 54 Brain glioblastoma POLE P436H 359 Pediatric New 55 Skin basal cell carcinoma POLE P286S 58.6 Adult New 56 Unknown primary melanoma POLE P286S 42.3 Adult New 57 Uterus endometrial POLE P286R 493.7 Adult Known adenocarcinoma (NOS) 58 Colon adenocarcinoma (CRC) POLE P286R 479.3 Adult Known 59 Brain glioblastoma (GBM) POLE P286R 450.5 Adult Known 60 Colon adenocarcinoma (CRC) POLE P286R 394.6 Adult Known 61 Colon adenocarcinoma (CRC) POLE P286R 342.3 Adult Known 62 Colon adenocarcinoma (CRC) POLE P286R 305.4 Adult Known 63 Colon adenocarcinoma (CRC) POLE P286R 259.5 Adult Known 64 Colon adenocarcinoma (CRC) POLE P286R 245 Adult Known 65 Brain glioblastoma (GBM) POLE P286R 227.9 Adult Known 66 Colon adenocarcinoma (CRC) POLE P286R 226.1 Adult Known 67 Rectum adenocarcinoma POLE P286R 223.4 Adult Known (CRC) 68 Colon adenocarcinoma (CRC) POLE P286R 216.2 Adult Known 69 Uterus endometrial POLE P286R 208.1 Adult Known adenocarcinoma (NOS) 70 Uterus endometrial POLE P286R 203.6 Adult Known adenocarcinoma (NOS) 71 Colon adenocarcinoma (CRC) POLE P286R 172.1 Adult Known 72 Uterus carcinosarcoma POLE P286R 168.5 Adult Known 73 Ovary neuroendocrine POLE P286R 163.1 Adult Known carcinoma 74 Rectum adenocarcinoma POLE P286R 162.2 Adult Known (CRC) 75 Colon adenocarcinoma (CRC) POLE P286R 155 Adult Known 76 Uterus endometrial POLE P286R 153.2 Adult Known adenocarcinoma (NOS) 77 Uterus endometrial POLE P286R 145.9 Adult Known adenocarcinoma (NOS) 78 Colon adenocarcinoma (CRC) POLE P286R 133.3 Adult Known 79 Uterus endometrial POLE P286R 123.4 Adult Known adenocarcinoma (NOS) 80 Pancreas ductal POLE P286R 100.9 Adult Known adenocarcinoma 81 Uterus endometrial POLE P286R 84.7 Adult Known adenocarcinoma (NOS) 82 rectum neuroendocrine POLE P286R 80.2 Adult Known carcinoma 83 Colon adenocarcinoma (CRC) POLE P286R 62.2 Adult Known 84 Brain glioblastoma (GBM) POLE P286H 212 Adult New 85 Brain glioblastoma (GBM) POLE M444K 116.2 Adult Known 86 Colon adenocarcinoma (CRC) POLE M444K 91.9 Adult Known 87 Uterus neuroendocrine POLE M444K 32.4 Adult Known carcinoma 88 Colorectal carcinoma POLE L424P 590 Pediatric New 89 Brain glioblastoma POLE L424I 336 Pediatric Known 90 Brain glioblastoma (GBM) POLE L424I 160.4 Adult Known 91 Lung adenocarcinoma POLE L424I 154.1 Adult Known 92 Liver cholangiocarcinoma POLE L424I 47.7 Adult Known 93 Brain glioblastoma POLE F367L 472.56 Pediatric New 94 Brain glioblastoma POLE F367L 238.18 Pediatric New 95 Unknown primary squamous POLE E978K 80.2 Adult New cell carcinoma (SCC) 96 Brain glioblastoma POLE E978G 608.02 Pediatric New Uterus carcinosarcoma POLE E978G 26.1 Adult New 98 Appendix adenocarcinoma POLE D368N 36 Adult New 99 Lung adenocarcinoma POLE D275V 43.2 Adult Known 100 Brain glioblastoma (GBM) POLE D275V 17.1 Adult Known 101 Pancreas ducta POLE D275G 318.9 Adult Known adenocarcinoma 102 Brain glioblastoma (GBM) POLE D275A 226.1 Adult Known 103 Rectum adenocarcinoma POLE C810G 854.1 Adult New (CRC) 104 Brain glioblastoma (GBM) POLE C810G 154.1 Adult New 105 Lung squamous cell POLE A463V 231.5 Adult New carcinoma (SCC) 106 Uterus endometrial POLE A463V 55.9 Adult New adenocarcinoma (NOS) 107 Brain oligodendroglioma POLE A463T 809 Adult New 108 Brain glioblastoma POLE A463D 532 Pediatric New 109 Brain glioblastoma POLE A463D 359 Pediatric New 110 Colon adenocarcinoma (CRC) POLE A456P 373.9 Adult Known 111 Colon adenocarcinoma (CRC) POLE A456P 314.4 Adult Known 112 Colon adenocarcinoma (CRC) POLE A456P 233.3 Adult Known 113 Uterus endometrial POLE A456P 226.1 Adult Known adenocarcinoma (NOS) 114 Colon adenocarcinoma (CRC) POLE A456P 200 Adult Known 115 Ovary endometrioid POLE A456P 69.4 Adult adenocarcinoma Known 116 Cervix squamous cel POLE A456P 33.3 Adult Known carcinoma (SCC) 117 Brain glioblastoma (GBM) POLE A288V 578.4 Adult New 118 Brain glioblastoma (GBM) POLE A288V 386.5 Adult New 119 Stomach neuroendocrine POLE A288V 155.9 Adult New carcinoma Drivers POLD1 1 Colon adenocarcinoma (CRC) POLD1 R689W 259.5 Adult Known 2 Brain oligodendroglioma POLD1 R689W 585.6 Adult Known 3 Brain glioblastoma (GBM) POLD1 R689W 578.4 Adult Known 4 Brain glioblastoma (GBM) POLD1 R689W 447.8 Adult Known 5 Colon adenocarcinoma (CRC) POLD1 R689W 12.6 Adult Known 6 Vulva squamous cell POLD1 R1016H 39.6 Adult New carcinoma (SCC) 7 Colon adenocarcinoma (CRC) POLD1 R1016H 29.7 Adult New 8 Brain glioma (NOS) POLD1 R1016H 315.3 Adult New 9 Unknown primary POLD1 R1016H 69.4 Adult New adenocarcinoma 10 Uterus endometrial POLD1 R1016H 636.9 Adult New adenocarcinoma (NOS) 11 Colon adenocarcinoma (CRC) POLD1 R1016H 49.5 Adult New 12 Brain medullobastoma POLD1 L632M 141 Pediatric New 13 Brain glioblastoma POLD1 L632M 228 Pediatric New 14 Brain glioblastoma POLD1 L632M 296 Pediatric New 15 Brain Glioblastoma POLD1 L632M 121 Pediatric New 16 Brain PNET POLD1 L606M 196 Pediatric Known 17 Brain PNET POLD1 L606M 295 Pediatric Known 18 Brain glioblastoma (GBM) POLD1 L606M 228.8 Adult Known 19 Brain glioblastoma (GBM) POLD1 L606M 51.4 Adult Known 20 Uterus carcinosarcoma POLD1 L606M 311.7 Adult Known 21 Brain PNET POLD1 E318K 182 Pediatric New 22 Lung non-small cell lung carcinoma (NOS) POLD1 E318K 13.5 Adult New 23 Colon adenocarcinoma (CRC) POLD1 E245K 76.6 Adult New 24 Liver cholangiocarcinoma POLD1 E245K 122.5 Adult New 25 Colon adenocarcinoma (CRC) POLD1 E245K 85.6 Adult New.


6. The method of claim 1, wherein the tumour is a hypermutant tumour.
 7. The method of claim 6, wherein the hypermutant tumour has mutation frequency of at least 5 mutations per megabase (Mb), and the tumour is a pediatric tumour.
 8. The method of claim 6, wherein the hypermutant tumour has a mutation frequency of at least 9.9 mutations per megabase (Mb), and the tumour is from an adult.
 9. The method of claim 6, wherein the hypermutant tumour is an ultra-hypermutant tumour having a mutation frequency of at least 100 mutations per megabase (Mb).
 10. The method of claim 2, wherein the step of assigning is carried out by matching the relative proportions determined for the tumour to one of the clusters by highest cosine similarity.
 11. The method of claim 10, wherein the matching requires a minimum cosine similarity of 0.75. 